Substituted bridged urea analogs as sirtuin modulators

ABSTRACT

Provided herein are novel substituted bridged urea and related analogs and methods of use thereof. The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders that would benefit from increased mitochondrial activity. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

BACKGROUND

The Silent Information Regulator (SIR) family of genes represents ahighly conserved group of genes present in the genomes of organismsranging from archaeabacteria to eukaryotes. The encoded SIR proteins areinvolved in diverse processes from regulation of gene silencing to DNArepair. A well-characterized gene in this family is S. cerevisiae SIR2,which is involved in silencing HM loci that contain informationspecifying yeast mating type, telomere position effects and cell aging.The yeast Sir2 protein belongs to a family of histone deacetylases. Theproteins encoded by members of the SIR gene family show high sequenceconservation in a 250 amino acid core domain. The Sir2 homolog, CobB, inSalmonella typhimurium, functions as an NAD (nicotinamide adeninedinucleotide)-dependent ADP-ribosyl transferase.

The Sir2 protein is a class III deacetylase which uses NAD as acosubstrate. Unlike other deacetylases, many of which are involved ingene silencing, Sir2 is insensitive to class I and II histonedeacetylase inhibitors like trichostatin A (TSA).

Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NADhydrolysis, producing nicotinamide and a novel acetyl-ADP ribosecompound. The NAD-dependent deacetylase activity of Sir2 is essentialfor its functions, which can connect its biological role with cellularmetabolism in yeast. Mammalian Sir2 homologs have NAD-dependent histonedeacetylase activity.

Biochemical studies have shown that Sir2 can readily deacetylate theamino-terminal tails of histones H3 and H4, resulting in the formationof 2′/3′-O-acetyl-ADP-ribose (OAADPR) and nicotinamide. Strains withadditional copies of SIR2 display increased rDNA silencing and a 30%longer life span. It has also been shown that additional copies of theC. elegans SIR2 homolog, sir-2.1, and the D. melanogaster dSir2 geneextend life span in those organisms. This implies that theSIR2-dependent regulatory pathway for aging arose early in evolution andhas been well conserved. Today, Sir2 genes are believed to have evolvedto enhance an organism's health and stress resistance to increase itschance of surviving adversity.

In humans, there are seven Sir2-like genes (SIRT1-SIRT7) that share theconserved catalytic domain of Sir2. SIRT1 is a nuclear protein with thehighest degree of sequence similarity to Sir2. SIRT1 regulates multiplecellular targets by deacetylation including the tumor suppressor p53,the cellular signaling factor NF-κB, and the FOXO transcription factor.

SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes andeukaryotes. The SIRT3 protein is targeted to the mitochondrial cristaeby a unique domain located at the N-terminus. SIRT3 has NAD⁺-dependentprotein deacetylase activity and is ubiquitously expressed, particularlyin metabolically active tissues. Upon transfer to the mitochondria,SIRT3 is believed to be cleaved into a smaller, active form by amitochondrial matrix processing peptidase (MPP).

Caloric restriction has been known for over 70 years to improve thehealth and extend the lifespan of mammals. Yeast life span, like that ofmetazoans, is also extended by interventions that resemble caloricrestriction, such as low glucose. The discovery that both yeast andflies lacking the SIR2 gene do not live longer when caloricallyrestricted provides evidence that SIR2 genes mediate the beneficialhealth effects of a restricted calorie diet. Moreover, mutations thatreduce the activity of the yeast glucose-responsive cAMP (adenosine3′,5′-monophosphate)-dependent (PKA) pathway extend life span in wildtype cells but not in mutant sir2 strains, demonstrating that SIR2 islikely to be a key downstream component of the caloric restrictionpathway.

In addition to therapeutic potential, structural and biophysical studiesof SIRT1 activity and activation by small molecule sirtuin modulatorswould be useful to advance understanding of the biological function ofsirtuins, to further the understanding of the mechanism of action ofsirtuin activation and to aid in the development of assays that identifynovel sirtuin modulators.

SUMMARY

Provided herein are novel sirtuin-modulating compounds and methods ofuse thereof.

In one aspect, the invention provides sirtuin-modulating compounds ofStructural Formulas (I), (IIa), (IIb), (IIIa), (IIIb) and (IV) as aredescribed in detail below.

In another aspect, the invention provides methods for usingsirtuin-modulating compounds, or compositions comprisingsirtuin-modulating compounds. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be used for a variety of therapeutic applications including, forexample, increasing the lifespan of a cell, and treating and/orpreventing a wide variety of diseases and disorders including, forexample, diseases or disorders related to aging or stress, diabetes,obesity, neurodegenerative diseases, chemotherapeutic-inducedneuropathy, neuropathy associated with an ischemic event, oculardiseases and/or disorders, cardiovascular disease, blood clottingdisorders, inflammation, and/or flushing, etc. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay also be used for treating a disease or disorder in a subject thatwould benefit from increased mitochondrial activity, for enhancingmuscle performance, for increasing muscle ATP levels, or for treating orpreventing muscle tissue damage associated with hypoxia or ischemia. Inother embodiments, sirtuin-modulating compounds that decrease the leveland/or activity of a sirtuin protein may be used for a variety oftherapeutic applications including, for example, increasing cellularsensitivity to stress, increasing apoptosis, treatment of cancer,stimulation of appetite, and/or stimulation of weight gain, etc. Asdescribed further below, the methods comprise administering to a subjectin need thereof a pharmaceutically effective amount of asirtuin-modulating compound.

In certain aspects, the sirtuin-modulating compounds may be administeredalone or in combination with other compounds, including othersirtuin-modulating compounds, or other therapeutic agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows ¹H-NMR spectrum of Compound 1.

FIG. 2 shows ¹³C-NMR and APT NMR spectrum of Compound 1.

FIG. 3 depicts (A) HDX-MS of full length SIRT1, (B) Enzymaticcharacterization of effect of CBS on SIRT1cc activity, (C) Pivot plotthe STAC activation of mini-hSIRT1(ΔN) vs. mini-hSIRT1, (D) Pivot plotthe STAC activation of mini-hSIRT1(ΔCBS) vs. mini-hSIRT1 and (E) Pivotplot the STAC activation of mini-hSIRT1(E230K) vs. mini-hSIRT1.

FIG. 4 shows chemical structures of synthetic SIRT1 activators (1, 4-9),inhibitor (2), and fluorescent polarization assay probe (3).

FIG. 5 depicts size exclusion chromatography (SEC) of mini-hSIRT1 in theabsence or presence of STACs.

FIG. 6 depicts (A) Differential perturbation of the HDX-MS profile ofSIRT1cc upon binding to CBS peptide (B) Structural comparison ofMini-hSIRT1/1 complex and ySIR2 (C) Structural comparison of theN-terminal SBD of Mini-hSIRT1/1 complex, Mini-hSIRT1/1/2 complex andMini-hSIRT1/1/Ac-p53-7mer/CarbaNAD quaternary complex.

FIG. 7 depicts the activation dose-response curves comparing wild-typeand (A) I223A or (B) E230K SIRT1 using the OAcADPr assay with theAc-p53(W5) substrate.

FIG. 8 depicts activation comparison of wild-type versus mutantfull-length hSIRT1.

FIG. 9 depicts (A) Interface of Mini-hSIRT1/Ac-p53 interaction. (B)Interface of Mini-hSIRT1/carbaNAD interaction. (C) Interface ofMini-hSIRT1/2 interaction.

FIG. 10 depicts (A) Binding of FP probe 3 to SIRT1. (B) Competition of 4against SIRT1/3 complex.

FIG. 11 depicts impaired STAC binding by full-length I223R hSIRT1.

FIG. 12 depicts pivot plot the STAC activation of mini-hSIRT1(R446A) vs.mini-hSIRT1.

DETAILED DESCRIPTION 1. Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues.

The term “bioavailable”, when referring to a compound, is art-recognizedand refers to a form of a compound that allows for all or a portion ofthe amount of compound administered to be absorbed by, incorporatedinto, or otherwise physiologically available to a subject or patient towhom it is administered.

“Biologically active portion of a sirtuin” refers to a portion of asirtuin protein having a biological activity, such as the ability todeacetylate (“catalytically active”). Catalytically active portions of asirtuin may comprise the core domain of sirtuins. Catalytically activeportions of SIRT1 having GenBank Accession No. NP_036370 that encompassthe NAD⁺ binding domain and the substrate binding domain, for example,may include without limitation, amino acids 240-664 or 240-505 ofGenBank Accession No. NP_036370, which are encoded by the polynucleotideof GenBank Accession No. NM_012238. Therefore, this region is sometimesreferred to as the core domain. Other catalytically active portions ofSIRT1, also sometimes referred to as core domains, include about aminoacids 261 to 447 of GenBank Accession No. NP_036370, which are encodedby nucleotides 834 to 1394 of GenBank Accession No. NM_012238; aboutamino acids 242 to 493 of GenBank Accession No. NP_036370, which areencoded by nucleotides 777 to 1532 of GenBank Accession No. NM_012238;or about amino acids 254 to 495 of GenBank Accession No. NP_036370,which are encoded by nucleotides 813 to 1538 of GenBank Accession No.NM_012238. Another “biologically active” portion of SIRT1 is amino acids62-293 or 183-225 of GenBank Accession No. NP_036370, which comprise adomain N-terminal to the core domain that is important to the compoundbinding site.

The term “companion animals” refers to cats and dogs. As used herein,the term “dog(s)” denotes any member of the species Canis familiaris, ofwhich there are a large number of different breeds. The term “cat(s)”refers to a feline animal including domestic cats and other members ofthe family Felidae, genus Felis.

“Diabetes” refers to high blood sugar or ketoacidosis, as well aschronic, general metabolic abnormalities arising from a prolonged highblood sugar status or a decrease in glucose tolerance. “Diabetes”encompasses both the type I and type II (Non Insulin Dependent DiabetesMellitus or NIDDM) forms of the disease. The risk factors for diabetesinclude the following factors: waistline of more than 40 inches for menor 35 inches for women, blood pressure of 130/85 mmHg or higher,triglycerides above 150 mg/dl, fasting blood glucose greater than 100mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50mg/dl in women.

The term “ED₅₀” refers to the art-recognized measure of effective dose.In certain embodiments, ED₅₀ means the dose of a drug which produces 50%of its maximum response or effect, or alternatively, the dose whichproduces a pre-determined response in 50% of test subjects orpreparations, such as isolated tissue or cells. The term “LD₅₀” refersto the art-recognized measure of lethal dose. In certain embodiments,LD₅₀ means the dose of a drug which is lethal in 50% of test subjects.The term “therapeutic index” is an art-recognized term which refers tothe therapeutic index of a drug, defined as LD₅₀/ED₅₀.

The term “hyperinsulinemia” refers to a state in an individual in whichthe level of insulin in the blood is higher than normal.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

An “insulin resistance disorder,” as discussed herein, refers to anydisease or condition that is caused by or contributed to by insulinresistance. Examples include: diabetes, obesity, metabolic syndrome,insulin-resistance syndromes, syndrome X, insulin resistance, high bloodpressure, hypertension, high blood cholesterol, dyslipidemia,hyperlipidemia, atherosclerotic disease including stroke, coronaryartery disease or myocardial infarction, hyperglycemia, hyperinsulinemiaand/or hyperproinsulinemia, impaired glucose tolerance, delayed insulinrelease, diabetic complications, including coronary heart disease,angina pectoris, congestive heart failure, stroke, cognitive functionsin dementia, retinopathy, peripheral neuropathy, nephropathy,glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensivenephrosclerosis, some types of cancer (such as endometrial, breast,prostate, and colon), complications of pregnancy, poor femalereproductive health (such as menstrual irregularities, infertility,irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy,cholesterol-related disorders, such as gallstones, cholecystitis andcholelithiasis, gout, obstructive sleep apnea and respiratory problems,osteoarthritis, and bone loss, e.g., osteoporosis in particular.

The term “livestock animals” refers to domesticated quadrupeds, whichincludes those being raised for meat and various byproducts, e.g., abovine animal including cattle and other members of the genus Bos, aporcine animal including domestic swine and other members of the genusSus, an ovine animal including sheep and other members of the genusOvis, domestic goats and other members of the genus Capra; domesticatedquadrupeds being raised for specialized tasks such as use as a beast ofburden, e.g., an equine animal including domestic horses and othermembers of the family Equidae, genus Equus.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, livestock animals (including bovines, porcines, etc.),companion animals (e.g., canines, felines, etc.) and rodents (e.g., miceand rats).

“Obese” individuals or individuals suffering from obesity are generallyindividuals having a body mass index (BMI) of at least 25 or greater.Obesity may or may not be associated with insulin resistance.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

A “patient”, “subject”, “individual” or “host” refers to either a humanor a non-human animal.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. If it is administeredprior to clinical manifestation of the unwanted condition (e.g., diseaseor other unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if administered after manifestation of the unwantedcondition, the treatment is therapeutic (i.e., it is intended todiminish, ameliorate or maintain the existing unwanted condition or sideeffects therefrom).

The term “pyrogen-free”, with reference to a composition, refers to acomposition that does not contain a pyrogen in an amount that would leadto an adverse effect (e.g., irritation, fever, inflammation, diarrhea,respiratory distress, endotoxic shock, etc.) in a subject to which thecomposition has been administered. For example, the term is meant toencompass compositions that are free of, or substantially free of, anendotoxin such as, for example, a lipopolysaccharide (LPS).

“Replicative lifespan” of a cell refers to the number of daughter cellsproduced by an individual “mother cell.” “Chronological aging” or“chronological lifespan,” on the other hand, refers to the length oftime a population of non-dividing cells remains viable when deprived ofnutrients. “Increasing the lifespan of a cell” or “extending thelifespan of a cell,” as applied to cells or organisms, refers toincreasing the number of daughter cells produced by one cell; increasingthe ability of cells or organisms to cope with stresses and combatdamage, e.g., to DNA, proteins; and/or increasing the ability of cellsor organisms to survive and exist in a living state for longer under aparticular condition, e.g., stress (for example, heatshock, osmoticstress, high energy radiation, chemically-induced stress, DNA damage,inadequate salt level, inadequate nitrogen level, or inadequate nutrientlevel). Lifespan can be increased by at least about 10%, 20%, 30%, 40%,50%, 60% or between 20% and 70%, 30% and 60%, 40% and 60% or more usingmethods described herein.

“Sirtuin-modulating compound” refers to a compound that increases thelevel of a sirtuin protein and/or increases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-modulatingcompound may increase at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

proteins include deacetylation, e.g., of an acetylated peptidesubstrate.

“Sirtuin protein” refers to a member of the sirtuin deacetylase proteinfamily, or preferably to the sir2 family, which include yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_501912), and human SIRT1 (GenBank Accession No. NM_012238 andNP_036370 (or AF083106)) and SIRT2 (GenBank Accession No. NM_012237,NM_030593, NP_036369, NP_085096, and AF083107) proteins. Other familymembers include the four additional yeast Sir2-like genes termed “HSTgenes” (homologues of Sir two) HST1, HST2, HST3 and HST4, and the fiveother human homologues hSIRT3, hSIRT4, hSIRT5, hSIRT6 and hSIRT7(Brachmann et al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC260:273).

“SIRT1 protein” refers to a member of the sir2 family of sirtuindeacetylases. In certain embodiments, a SIRT1 protein includes yeastSir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBankAccession No. NP_501912), human SIRT1 (GenBank Accession No. NM_012238or NP_036370 (or AF083106)), mouse SIRT1 (GenBank Accession No.NM_019812 or NP_062786), and equivalents and fragments thereof. Inanother embodiment, a SIRT1 protein includes a polypeptide comprising asequence consisting of, or consisting essentially of, the amino acidsequence set forth in GenBank Accession Nos. NP_036370, NP_501912,NP_085096, NP_036369, or P53685. SIRT1 proteins include polypeptidescomprising all or a portion of the amino acid sequence set forth inGenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, orP53685; the amino acid sequence set forth in GenBank Accession Nos.NP_036370, NP_501912, NP_085096, NP_036369, or P53685 with 1 to about 2,3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acidsubstitutions; an amino acid sequence that is at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.NP_036370, NP_501912, NP_085096, NP_036369, or P53685, and functionalfragments thereof. Polypeptides of the invention also include homologs(e.g., orthologs and paralogs), variants, or fragments, of GenBankAccession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685.

As used herein “SIRT2 protein”, “SIRT3 protein”, “SIRT4 protein”, SIRT5protein”, “SIRT6 protein”, and “SIRT7 protein” refer to other mammalian,e.g. human, sirtuin deacetylase proteins that are homologous to SIRT1protein, particularly in the approximately 275 amino acid conservedcatalytic domain. For example, “SIRT3 protein” refers to a member of thesirtuin deacetylase protein family that is homologous to SIRT1 protein.In certain embodiments, a SIRT3 protein includes human SIRT3 (GenBankAccession No. AAH01042, NP_036371, or NP_001017524) and mouse SIRT3(GenBank Accession No. NP_071878) proteins, and equivalents andfragments thereof. In certain embodiments, a SIRT4 protein includeshuman SIRT4 (GenBank Accession No. NM_012240 or NP_036372). In certainembodiments, a SIRT5 protein includes human SIRT5 (GenBank Accession No.NM_012241 or NP_036373). In certain embodiments, a SIRT6 proteinincludes human SIRT6 (GenBank Accession No. NM_016539 or NP 057623). Inanother embodiment, a SIRT3 protein includes a polypeptide comprising asequence consisting of, or consisting essentially of, the amino acidsequence set forth in GenBank Accession Nos. AAH01042, NP_036371,NP_001017524, or NP_071878. SIRT3 proteins include polypeptidescomprising all or a portion of the amino acid sequence set forth inGenBank Accession AAH01042, NP_036371, NP_001017524, or NP_071878; theamino acid sequence set forth in GenBank Accession Nos. AAH01042,NP_036371, NP_001017524, or NP_071878 with 1 to about 2, 3, 5, 7, 10,15, 20, 30, 50, 75 or more conservative amino acid substitutions; anamino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, or 99% identical to GenBank Accession Nos. AAH01042, NP_036371,NP_001017524, or NP_071878, and functional fragments thereof.Polypeptides of the invention also include homologs (e.g., orthologs andparalogs), variants, or fragments, of GenBank Accession Nos. AAH01042,NP_036371, NP_001017524, or NP_071878. In certain embodiments, a SIRT3protein includes a fragment of SIRT3 protein that is produced bycleavage with a mitochondrial matrix processing peptidase (MPP) and/or amitochondrial intermediate peptidase (MIP).

The term “stereoisomer” as used herein is art-recognized and refers toany of two or more isomers that have the same molecular constitution anddiffer only in the three-dimensional arrangement of their atomicgroupings in space. When used herein to describe a compounds or genus ofcompounds, stereoisomer includes any portion of the compound or thecompound in its entirety. For example, diastereomers and enantiomers arestereoisomers.

The terms “systemic administration” and “administered systemically,” areart-recognized and refer to the administration of a subject composition,therapeutic or other material enterally or parenterally.

The term “tautomer” as used herein is art-recognized and refers to anyone of the possible alternative structures that may exist as a result oftautomerism, which refers to a form of constitutional isomerism in whicha structure may exist in two or more constitutional arrangements,particularly with respect to the position of hydrogens bonded to oxygen.When used herein to describe a compound or genus of compounds, it isfurther understood that a “tautomer” is readily interconvertible andexists in equilibrium. For example, keto and enol tautomers exist inproportions determined by the equilibrium position for any givencondition, or set of conditions:

The term “therapeutic agent” is art-recognized and refers to anybiologically, physiologically, or pharmacologically active substancethat acts locally or systemically in a subject. The term also means anysubstance intended for use in the diagnosis, cure, mitigation, treatmentor prevention of disease or in the enhancement of desirable physical ormental development and/or conditions in an animal or human.

The term “therapeutic effect” is art-recognized and refers to abeneficial local or systemic effect in animals, particularly mammals,and more particularly humans, caused by a pharmacologically activesubstance. The phrase “therapeutically-effective amount” means thatamount of such a substance that produces some desired local or systemiceffect at a reasonable benefit/risk ratio applicable to any treatment.The therapeutically effective amount of such substance will varydepending upon the subject and disease condition being treated, theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of skill in the art. For example, certain compositionsdescribed herein may be administered in a sufficient amount to produce adesired effect at a reasonable benefit/risk ratio applicable to suchtreatment.

“Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease.

The term “vision impairment” refers to diminished vision, which is oftenonly partially reversible or irreversible upon treatment (e.g.,surgery). Particularly severe vision impairment is termed “blindness” or“vision loss”, which refers to a complete loss of vision, vision worsethan 20/200 that cannot be improved with corrective lenses, or a visualfield of less than 20 degrees diameter (10 degrees radius).

2. Compounds

In one aspect, the invention provides novel compounds for treatingand/or preventing a wide variety of diseases and disorders including,for example, diseases or disorders related to aging or stress, diabetes,obesity, neurodegenerative diseases, ocular diseases and disorders,cardiovascular disease, blood clotting disorders, inflammation, cancer,and/or flushing, etc. Subject compounds, such as sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin protein,may also be used for treating a disease or disorder in a subject thatwould benefit from increased mitochondrial activity, for enhancingmuscle performance, for increasing muscle ATP levels, or for treating orpreventing muscle tissue damage associated with hypoxia or ischemia.Compounds disclosed herein may be suitable for use in pharmaceuticalcompositions and/or one or more methods disclosed herein.

In certain embodiments, compounds of the invention are represented byStructural Formula (I):

The invention includes pharmaceutical compositions of any of thecompounds of Structural Formulas (I), (IIa), (IIb), (IIIa), (IIIb), and(IV) or as otherwise set forth above. The pharmaceutical composition ofthe compound of Structural Formulas I), (IIa), (IIb), (IIIa), (IIIb),and (IV) may comprise one or more pharmaceutically acceptable carriersor diluents.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Structural Formula (I):

or a salt thereof wherein:

m is 1 or 2;

n is 2 or 3;

p is 0 to 4;

R¹ is selected from a carbocycle and heterocycle, wherein R¹ isoptionally substituted with one or more substituents independentlyselected from halo, C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, —C≡N,—Y, —X—C(═O)—Y, —X—O—Y, —X—OR⁴, —X—C(═O)—NR³R³, —X—NH—C(═O)—Y—NR³R³,—X—NH—C(═O)—O—Y, —X—NR³R³, ═O, —NH—S(═O)₂—R₃, —S(═O)₂—R³, —S—R³,—(C₃-C₇) cycloalkyl, —C(═N)—NR³R³, —C(═N)—NH—X—NR³R³, —X—NH—C(═O)—Y,—C(═O)—NH—X, —NH—X, phenyl, —O-phenyl, 3- to 6-membered saturated orunsaturated heterocycle and —O-(5- to 6-membered saturated heterocycle),wherein any phenyl, 3- to 6-membered saturated or unsaturatedheterocycle or —O-5- to 6-membered saturated heterocycle substituent ofR¹ is optionally substituted at any substitutable carbon atom with oneor more substituents selected from halo, —OR⁴, —X—O—Y, —CF₃, —Y,—X—R³R³, —X—NH—C(═O)—Y—NR³R³, —X—NH—C(═O)—O—Y-(5- to 6-memberedsaturated heterocycle or carbocycle), —X—C(═N)—NR³R³ and —S—Y andoptionally substituted at any substitutable nitrogen atom with —Y,—C(═O)—Y, —C(═O)—O—Y, —C(═O)—OR⁴, —Y—C(═O)—Y—NR³R³, —Y—NH—C(═O)—O—Y,—Y—NH—C(═O)—OR⁴, —Y—NH₂, —C(═O)—NH—Y or —C(═O)-3- to 5-memberedsaturated carbocycle;

R² is selected from a carbocycle and a heterocycle, wherein R² isoptionally substituted with one or more substituents independentlyselected from halo, C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, —C≡N,—Y, —X—OR⁴, —X—O—Y, —SO₂—R³, —X—NR³R³, —NH—S(═O)₂R³, —C(═O)—NR³R³,—C(═O)—Y, —C(═O)—O—Y, —SO₂—R_(y), —SO₂—NH—R_(y), —SO₂—NR³R³, 3- to6-membered saturated carbocycle or heterocycle and phenyl, wherein any3- to 6-membered saturated heterocycle substituent of R² is optionallysubstituted at any carbon atom with one or more substituents selectedfrom halo, —CF₃, —Y, —X—O—Y, —NH—Y and —N(Y)₂, and is optionallysubstituted at any nitrogen atom with one or more substituents selectedfrom —C(═O)—O—Y, —Y and —C(═O)—Y, and when R² is an N-linked 5- to7-membered saturated or unsaturated heterocycle it is furthersubstituted at any nitrogen atom with one or more substituents selectedfrom —C(═O)—O—Y, —Y and —C(═O)—Y;

each R³ is independently selected from hydrogen, —C(═N)—NH₂, —C(═O)—Y,—Y, —Y—NH—C(═O)—O—Y, —Y—NH—C(═O)—OH, —Y—NH—C(═O)—CF₃, —C(═O)—Y-3- to5-membered saturated heterocycle, —C(═O)—O—Y-(3- to 5-membered saturatedheterocycle), —C(═O)—CF₃, —C(═O)—O—Y, —C(═O)—OH, —C(═O)—O—CF₃,—S(═O)₂—Y, —S(═O)₂—OH;

two R³ are taken together with the nitrogen or carbon atom to which theyare bound to form a 4- to 8-membered saturated heterocycle optionallycomprising one additional heteroatom selected independently from N, S,S(═O), S(═O)₂, and O, wherein the heterocycle formed by two R³ isoptionally substituted at any carbon atom with one or more of OH, halo,Y, NH₂, NH—Y, N(Y)₂, O—Y, and optionally substituted at anysubstitutable nitrogen atom with C(═O)—O—Y, Y or C(═O)—Y;

each R⁴ is independently selected from hydrogen, Y, —CF₃, —C(═O)—Y,—C(═O)—O—Y, —Y—C(═O)—Y or —Y—C(═O)—O—Y;

R⁵ and R⁶ are independently selected from hydrogen, —OH, —OCF₃, —O—Y,—O—C(═O)—Y, —O—C(═O)—O—Y, —O—C(═O)—NH—Y, —O—C(═O)—N(Y)₂, —O—C(═O)-5- to6-membered saturated or unsaturated heterocycle or carbocycle, whereinonly one of R⁵ and R⁶ is O—C(═O)-5- to 6-membered saturated orunsaturated heterocycle or carbocycle, and when R⁵ or R⁶ is O—C(═O)-5-to 6-membered saturated or unsaturated heterocycle or carbocycle it isfurther substituted with halo, —OH, Y, —O—Y, —OCF₃ or —O—C(═O)—Y; or

R⁵ and R⁶ can be taken together to the carbon atom to which they arebound to form ═O;

R⁷ and R⁸ are independently selected from hydrogen, halo, —OH, —O—Y andY;

R⁹ is selected from hydrogen, halo, —OH, —OCF₃, —O—Y, Y, —O—C(═O)—Y,—NH—Y and —N(Y)₂;

each X is C₀-C₅ straight chain or branched alkyl, alkenyl or alkynyl;and

each Y is C₁-C₅ straight chain or branched alkyl, alkenyl or alkynyl;

wherein any Y or X is optionally substituted with one or more of —OH,—C₁-C₄ straight chain or branched alkyl, —C₁-C₄ alkene, —C₁-C₄ alkynyl,—O—(C₁-C₄ alkyl), —O—(C₁-C₄ alkene), —O—(C₁-C₄ alkynyl), —C(═O)—C₁-C₄straight chain or branched alkyl, —C(═O)—C₁-C₄ alkene, —C(═O)—C₁-C₄alkynyl, —C(═O)—O—C₁-C₄ straight chain or branched alkyl, —C(═O)—O—C₁-C₄alkene, —C(═O)—O—C₁-C₄ alkynyl, halo, —NH₂, —NH(C₁-C₄ alkyl), —N(C₁-C₄alkyl)₂, —(C₁-C₃ straight chain or branched alkyl)-NH—(═NH)—NH₂,—NH(alkoxy-substituted C₁-C₄ alkyl), —NH(hydroxy-substituted C₁-C₄alkyl), —N(alkoxy-substituted C₁-C₄ alkyl)(hydroxy-substituted C₁-C₄alkyl), —N(hydroxy-substituted C₁-C₄ alkyl)₂ or —N(alkoxy-substitutedC₁-C₄ alkyl)₂.

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by the structure represented by Structural Formula (IIa):

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by the structure represented by Structural Formula (IIIa):

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by the structure represented by Structural Formulas (IIb)or (IIIb):

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by the structure represented by Structural Formula (IV):

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by having a p=0 (i.e., no methylene between the C(O)—NHand R₁).

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by having a p=1-4 (i.e., 1 to 4 methylenes between theC(O)—NH and R₁).

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by having a —(CH₂)p-R¹ group selected from:

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by an R¹ group that is phenyl, a saturated or unsaturated5- to 6-membered heterocycle, or a fused bicyclic 8- to 11-memberedsaturated or unsaturated carbocycle or heterocycle.

In particular embodiments, compounds or salts of Structural Formula (I)are characterized by an R¹ that is a fused bicyclic 8- to 11-memberedsaturated or unsaturated heterocycle.

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by an R¹ group that is selected from any one of:

In particular embodiments, compounds or salts of Structural Formula (I)are characterized by an R¹ group that is selected from any one of:

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by an R² group that is selected from a 5- to 7-memberedsaturated carbocycle or heterocycle, an N-linked heterocycle, and an 8-to 11-membered saturated or unsaturated heterocycle.

In particular embodiments, compounds or salts of Structural Formula (I)are characterized by an R² group that is an N-linked 5- to 7-memberedsaturated or unsaturated heterocycle.

In certain embodiments of the above, the compound is selected from anyone of:

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by an R² group that is a fused bicyclic 8- to 11-memberedsaturated or unsaturated heterocycle.

In certain embodiments of the above, the compound is selected from anyone of:

In certain embodiments, compounds or salts of Structural Formula (I) arecharacterized by an R² group that is selected from any one of:

In particular embodiments, compounds or salts of Structural Formula (I)are characterized by an R² group that is selected from any one of:

In certain embodiments, compounds or salts of Structural Formula (I) areselected from any one of:

In certain embodiments, compounds or salts of Structural Formula (IIa)are selected from any one of:

In certain embodiments, compounds or salts of Structural Formula (Ma)are selected from any one of:

In certain embodiments, compounds or salts of Structural Formula (IIb)are selected from:

In certain embodiments, compounds or salts of Structural Formula (IIIb)are selected from:

In certain embodiments, compounds or salts of Structural Formula (IV)are selected from any one of:

Compounds of the invention, including novel compounds of the invention,can also be used in the pharmaceutical compositions and the methodsdescribed herein.

In any of the preceding embodiments, a C₁-C₄ alkoxy-substituted groupmay include one or more alkoxy substituents such as one, two or threemethoxy groups or a methoxy group and an ethoxy group, for example.Exemplary C₁-C₄ alkoxy substituents include methoxy, ethoxy, isopropoxy,and tert-butoxy.

In any of the preceding embodiments, a hydroxy-substituted group mayinclude one or more hydroxy substituents, such as two or three hydroxygroups.

In any of the preceding embodiments, a “halo-substituted” group includesfrom one halo substituent up to perhalo substitution. Exemplaryhalo-substituted C₁-C₄ alkyl includes CFH₂, CClH₂, CBrH₂, CF₂H, CCl₂H,CBr₂H, CF₃, CCl₃, CBr₃, CH₂CH₂F, CH₂CH₂Cl, CH₂CH₂Br, CH₂CHF₂, CHFCH₃,CHClCH₃, CHBrCH₃, CF₂CHF₂, CF₂CHCl₂, CF₂CHBr₂, CH(CF₃)₂, and C(CF₃)₃.Perhalo-substituted C₁-C₄ alkyl, for example, includes CF₃, CCl₃, CBr₃,CF₂CF₃, CCl₂CF₃ and CBr₂CF₃.

In any of the preceding embodiments, a “carbocycle” group may refer to amonocyclic carbocycle embodiment and/or a polycyclic carbocycleembodiment, such as a fused, bridged or bicyclic carbocycle embodiment.“Carbocycle” groups of the invention may further refer to an aromaticcarbocycle embodiment and/or a non-aromatic carbocycle embodiment, or,in the case of polycyclic embodiments, a carbocycle having both one ormore aromatic rings and/or one or more non-aromatic rings. Polycycliccarbocycle embodiments may be a bicyclic ring, a fused ring or a bridgedbicycle. Non-limiting exemplary carbocycles include phenyl, cyclohexane,cyclopentane, or cyclohexene, amantadine, cyclopentane, cyclohexane,bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene,adamantane, decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,norbornane, decalin, spiropentane, memantine, biperiden, rimantadine,camphor, cholesterol, 4-phenylcycicohexanol, bicyclo[4.2.0]octane,memantine and 4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene.

In any of the preceding embodiments, a “heterocycle” group may refer toa monocyclic heterocycle embodiment and/or a polycyclic heterocyclicembodiment, such as a fused, bridged or bicyclic heterocycle embodiment.“Heterocycle” groups of the invention may further refer to an aromaticheterocycle embodiment and/or a non-aromatic heterocycle embodiment, or,in the case of polycyclic embodiments, a heterocycle having both one ormore aromatic rings and/or one or more non-aromatic rings. Polycyclicheterocycle embodiments may be a bicyclic ring, a fused ring or abridged bicycle. Non-limiting exemplary heterocycles include pyridyl,pyrrolidine, piperidine, piperazine, pyrrolidine, morpholine,pyrimidine, benzofuran, indole, quinoline, lactones, lactams,benzodiazepine, indole, quinoline, purine, adenine, guanine,4,5,6,7-tetrahydrobenzo[d]thiazole, hexamine and methenamine.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, (R)- and(S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

The compounds and salts thereof described herein can also be present asthe corresponding hydrates (e.g., hemihydrate, monohydrate, dihydrate,trihydrate, tetrahydrate) or solvates. Suitable solvents for preparationof solvates and hydrates can generally be selected by a skilled artisan.

The compounds and salts thereof can be present in amorphous orcrystalline (including co-crystalline and polymorph) forms.

Sirtuin-modulating compounds of the invention advantageously modulatethe level and/or activity of a sirtuin protein, particularly thedeacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, certainsirtuin-modulating compounds of the invention do not substantially haveone or more of the following activities: inhibition of PI3-kinase,inhibition of aldoreductase, inhibition of tyrosine kinase,transactivation of EGFR tyrosine kinase, coronary dilation, orspasmolytic activity, at concentrations of the compound that areeffective for modulating the deacetylation activity of a sirtuin protein(e.g., such as a SIRT1 and/or a SIRT3 protein).

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₄ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

The terms “alkenyl” (“alkene”) and “alkynyl” (“alkyne”) refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyl groups described above, but that contain atleast one double or triple bond respectively.

The term “aromatic carbocycle” refers to an aromatic hydrocarbon ringsystem containing at least one aromatic ring. The ring may be fused orotherwise attached to other aromatic carbocyclic rings or non-aromaticcarbocyclic rings. Examples of aromatic carbocycle groups includecarbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl.

“Azabicyclo” refers to a bicyclic molecule that contains a nitrogen atomin the ring skeleton. The two rings of the bicycle may be fused at twomutually bonded atoms, e.g., indole, across a sequence of atoms, e.g.,azabicyclo[2.2.1]heptane, or joined at a single atom, e.g., spirocycle.

“Bicycle” or “bicyclic” refers to a two-ring system in which one, two orthree or more atoms are shared between the two rings. Bicycle includesfused bicycles in which two adjacent atoms are shared by each of the tworings, e.g., decalin, indole. Bicycle also includes spiro bicycles inwhich two rings share a single atom, e.g., spiro[2.2]pentane,1-oxa-6-azaspiro[3.4]octane. Bicycle further includes bridged bicyclesin which at least three atoms are shared between two rings, e.g.,norbornane.

“Bridged bicycle” compounds are bicyclic ring systems in which at leastthree atoms are shared by both rings of the system, i.e., they includeat least one bridge of one or more atoms connecting two bridgeheadatoms. Bridged azabicyclo refers to a bridged bicyclic molecule thatcontains a nitrogen atom in at least one of the rings.

The term “Boc” refers to a tert-butyloxycarbonyl group (a common amineprotecting group).

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from non-aromatic and aromatic rings.Carbocycle includes bicyclic molecules in which one, two or three ormore atoms are shared between the two rings. The term “fused carbocycle”refers to a bicyclic carbocycle in which each of the rings shares twoadjacent atoms with the other ring. Each ring of a fused carbocycle maybe selected from non-aromaticaromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a non-aromatic oraromatic ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of non-aromatic and aromatic bicyclic rings, as valencepermits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one ormore positions capable of bearing a hydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated (non-aromatic). Typically, a cycloalkyl group has from 3 toabout 10 carbon atoms, more typically 3 to 8 carbon atoms unlessotherwise defined. A “cycloalkenyl” group is a cyclic hydrocarboncontaining one or more double bonds.

A “halogen” designates F, Cl, Br or I.

A “halogen-substitution” or “halo” substitution designates replacementof one or more hydrogens with F, Cl, Br or I.

The term “heteroaryl” or “aromatic heterocycle” includes substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The term“heteroaryl” also includes ring systems having one or two rings whereinat least one of the rings is heteroaromatic, e.g., the other cyclicrings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aromaticcarbocycle, heteroaryl, and/or heterocyclyl. Heteroaryl groups include,for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

The terms “heterocycle”, and “heterocyclic”, as used herein, refers to anon-aromatic or aromatic ring comprising one or more heteroatomsselected from, for example, N, O, B and S atoms, preferably N, O, or S.The term “heterocycle” includes both “aromatic heterocycles” and“non-aromatic heterocycles.” Heterocycles include 4-7 memberedmonocyclic and 8-U membered bicyclic rings. Heterocycle includesbicyclic molecules in which one, two or three or more atoms are sharedbetween the two rings. Each ring of a bicyclic heterocycle may beselected from non-aromatic and aromatic rings. The term “fusedheterocycle” refers to a bicyclic heterocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedheterocycle may be selected from non-aromatic and aromatic rings. In anexemplary embodiment, an aromatic ring, e.g., pyridyl, may be fused to anon-aromatic or aromatic ring, e.g., cyclohexane, cyclopentane,pyrrolidine, 2,3-dihydrofuran or cyclohexene. “Heterocycle” groupsinclude, for example, piperidine, piperazine, pyrrolidine, morpholine,pyrimidine, benzofuran, indole, quinoline, lactones, and lactams.Exemplary “fused heterocycles” include benzodiazepine, indole,quinoline, purine, and 4,5,6,7-tetrahydrobenzo[d]thiazole.“Heterocycles” may be substituted at any one or more positions capableof bearing a hydrogen atom.

“Monocyclic rings” include 5-7 membered aromatic carbocycle orheteroaryl, 3-7 membered cycloalkyl or cycloalkenyl, and 5-7 memberednon-aromatic heterocyclyl. Exemplary monocyclic groups includesubstituted or unsubstituted heterocycles or carbocycles such asthiazolyl, oxazolyl, oxazinyl, thiazinyl, dithianyl, dioxanyl,isoxazolyl, isothiazolyl, triazolyl, furanyl, tetrahydrofuranyl,dihydrofuranyl, pyranyl, tetrazolyl, pyrazolyl, pyrazinyl, pyridazinyl,imidazolyl, pyridinyl, pyrrolyl, dihydropyrrolyl, pyrrolidinyl,piperidinyl, piperazinyl, pyrimidinyl, morpholinyl,tetrahydrothiophenyl, thiophenyl, cyclohexyl, cyclopentyl, cyclopropyl,cyclobutyl, cycloheptanyl, azetidinyl, oxetanyl, thiiranyl, oxiranyl,aziridinyl, and thiomorpholinyl.

As used herein, “substituted” means substituting a hydrogen atom in astructure with an atom or molecule other than hydrogen. A substitutableatom such as a “substitutable nitrogen” is an atom that bears a hydrogenatom in at least one resonance form. The hydrogen atom may besubstituted for another atom or group such as a CH₃ or an OH group. Forexample, the nitrogen in a piperidine molecule is substitutable if thenitrogen is bound to a hydrogen atom. If, for example, the nitrogen of apiperidine is bound to an atom other than hydrogen, the nitrogen is notsubstitutable. An atom that is not capable of bearing a hydrogen atom inany resonance form is not substitutable.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. As usedherein, the term “stable” refers to compounds that possess stabilitysufficient to allow manufacture and that maintain the integrity of thecompound for a sufficient period of time to be useful for the purposesdetailed herein.

The compounds disclosed herein also include partially and fullydeuterated variants. In certain embodiments, deuterated variants may beused for kinetic studies. One of skill in the art can select the sitesat which such deuterium atoms are present.

Also included in the present invention are salts, particularlypharmaceutically acceptable salts, of the compounds described herein.The compounds of the present invention that possess a sufficientlyacidic, a sufficiently basic, or both functional groups, can react withany of a number of inorganic bases, and inorganic and organic acids, toform a salt. Alternatively, compounds that are inherently charged, suchas those with quaternary nitrogen, can form a salt with an appropriatecounterion (e.g., a halide such as bromide, chloride, or fluoride,particularly bromide).

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

According to another embodiment, the present invention provides methodsof producing the above-defined compounds. The compounds may besynthesized using conventional techniques. Advantageously, thesecompounds are conveniently synthesized from readily available startingmaterials.

Synthetic chemistry transformations and methodologies useful insynthesizing the compounds described herein are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieser and M.Fieser, Fieser and Fieser's Reagents for Organic Synthesis (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (1995).

In an exemplary embodiment, a therapeutic compound may traverse thecytoplasmic membrane of a cell. For example, a compound may have acell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.

Compounds described herein may also have one or more of the followingcharacteristics: the compound may be essentially non-toxic to a cell orsubject; the compound may be an organic molecule or a small molecule of2000 amu or less, 1000 amu or less; a compound may have a half-lifeunder normal atmospheric conditions of at least about 30 days, 60 days,120 days, 6 months or 1 year; the compound may have a half-life insolution of at least about 30 days, 60 days, 120 days, 6 months or 1year; a compound may be more stable in solution than resveratrol by atleast a factor of about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 foldor 100 fold; a compound may promote deacetylation of the DNA repairfactor Ku70; a compound may promote deacetylation of RelA/p65; acompound may increase general turnover rates and enhance the sensitivityof cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to inhibit a histone deacetylase (HDAC) class I,and/or an HDAC class II at concentrations (e.g., in vivo) effective formodulating the deacetylase activity of the sirtuin. For instance, inpreferred embodiments, the sirtuin-modulating compound is asirtuin-modulating compound and is chosen to have an EC₅₀ for activatingsirtuin deacetylase activity that is at least 5 fold less than the EC₅₀for inhibition of an HDAC I and/or HDAC II, and even more preferably atleast 10 fold, 100 fold or even 1000 fold less. Methods for assayingHDAC I and/or HDAC II activity are well known in the art and kits toperform such assays may be purchased commercially. See e.g., BioVision,Inc. (Mountain View, Calif.; world wide web at biovision.com) and ThomasScientific (Swedesboro, N.J.; world wide web at tomassci.com).

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to modulate sirtuin homologs. In certainembodiments, an activator of a human sirtuin protein may not have anysubstantial ability to activate a sirtuin protein from lower eukaryotes,particularly yeast or human pathogens, at concentrations (e.g., in vivo)effective for activating the deacetylase activity of human sirtuin. Forexample, a sirtuin-modulating compound may be chosen to have an EC₅₀ foractivating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylaseactivity that is at least 5 fold less than the EC₅₀ for activating ayeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), andeven more preferably at least 10 fold, 100 fold or even 1000 fold less.In another embodiment, an inhibitor of a sirtuin protein from lowereukaryotes, particularly yeast or human pathogens, does not have anysubstantial ability to inhibit a sirtuin protein from humans atconcentrations (e.g., in vivo) effective for inhibiting the deacetylaseactivity of a sirtuin protein from a lower eukaryote. For example, asirtuin-inhibiting compound may be chosen to have an IC₅₀ for inhibitinga human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity thatis at least 5 fold less than the IC₅₀ for inhibiting a yeast sirtuin,such as Sir2 (such as Candida, S. cerevisiae, etc.), and even morepreferably at least 10 fold, 100 fold or even 1000 fold less.

In certain embodiments, a sirtuin-modulating compound may have theability to modulate one or more sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,or SIRT7. In some embodiments, a sirtuin-modulating compound has theability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT1. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT1deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In some embodiments, a SIRT1 modulator does not have anysubstantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT3 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT3. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT3deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In some embodiments, a SIRT3 modulator does not have anysubstantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin-modulating compound may have a bindingaffinity for a sirtuin protein of about 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M orless. A sirtuin-modulating compound may reduce (activator) or increase(inhibitor) the apparent Km of a sirtuin protein for its substrate orNAD⁺ (or other cofactor) by a factor of at least about 2, 3, 4, 5, 10,20, 30, 50 or 100. In certain embodiments, Km values are determinedusing the mass spectrometry assay described herein. Preferred activatingcompounds reduce the Km of a sirtuin for its substrate or cofactor to agreater extent than caused by resveratrol at a similar concentration orreduce the Km of a sirtuin for its substrate or cofactor similar to thatcaused by resveratrol at a lower concentration. A sirtuin-modulatingcompound may increase the Vmax of a sirtuin protein by a factor of atleast about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin-modulatingcompound may have an ED₅₀ for modulating the deacetylase activity of aSIRT1 and/or SIRT3 protein of less than about 1 nM, less than about 10nM, less than about 100 nM, less than about 1 μM, less than about 10 μM,less than about 100 μM, or from about 1-10 nM, from about 10-100 nM,from about 0.1-1 μM, from about 1-10 μM or from about 10-100 μM. Asirtuin-modulating compound may modulate the deacetylase activity of aSIRT1 and/or SIRT3 protein by a factor of at least about 5, 10, 20, 30,50, or 100, as measured in a cellular assay or in a cell based assay. Asirtuin-modulating compound may cause at least about 10%, 30%, 50%, 80%,2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater induction of thedeacetylase activity of a sirtuin protein relative to the sameconcentration of resveratrol. A sirtuin-modulating compound may have anED₅₀ for modulating SIRT5 that is at least about 10 fold, 20 fold, 30fold, 50 fold greater than that for modulating SIRT1 and/or SIRT3.

3. Exemplary Uses

In certain aspects, the invention provides methods for modulating thelevel and/or activity of a sirtuin protein and methods of use thereof.

In certain embodiments, the invention provides methods for usingsirtuin-modulating compounds wherein the sirtuin-modulating compoundsactivate a sirtuin protein, e.g., increase the level and/or activity ofa sirtuin protein. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for a variety oftherapeutic applications including, for example, increasing the lifespanof a cell, and treating and/or preventing a wide variety of diseases anddisorders including, for example, diseases or disorders related to agingor stress, diabetes, obesity, neurodegenerative diseases, cardiovasculardisease, blood clotting disorders, inflammation, cancer, and/orflushing, etc. The methods comprise administering to a subject in needthereof a pharmaceutically effective amount of a sirtuin-modulatingcompound, e.g., a sirtuin-modulating compound.

Without wishing to be bound by theory, it is believed that activators ofthe instant invention may interact with a sirtuin at the same locationwithin the sirtuin protein (e.g., active site or site affecting the Kmor Vmax of the active site). It is believed that this is the reason whycertain classes of sirtuin activators and inhibitors can havesubstantial structural similarity.

In certain embodiments, the sirtuin-modulating compounds describedherein may be taken alone or in combination with other compounds. Incertain embodiments, a mixture of two or more sirtuin-modulatingcompounds may be administered to a subject in need thereof. In anotherembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered with one ormore of the following compounds: resveratrol, butein, fisetin,piceatannol, or quercetin. In an exemplary embodiment, asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein may be administered in combination with nicotinic acidor nicotinamide riboside. In another embodiment, a sirtuin-modulatingcompound that decreases the level and/or activity of a sirtuin proteinmay be administered with one or more of the following compounds:nicotinamide (NAM), suramin; NF023 (a G-protein antagonist); NF279 (apurinergic receptor antagonist); Trolox(6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid);(−)-epigallocatechin (hydroxy on sites 3,5,7,3′,4′, 5′);(−)-epigallocatechin gallate (Hydroxy sites 5,7,3′,4′,5′ and gallateester on 3); cyanidin chloride (3,5,7,3′,4′-pentahydroxyflavyliumchloride); delphinidin chloride (3,5,7,3′,4′,5′-hexahydroxyflavyliumchloride); myricetin (cannabiscetin; 3,5,7,3′,4′,5′-hexahydroxyflavone);3,7,3′,4′,5′-pentahydroxyflavone; gossypetin(3,5,7,8,3′,4′-hexahydroxyflavone), sirtinol; and splitomicin. In yetanother embodiment, one or more sirtuin-modulating compounds may beadministered with one or more therapeutic agents for the treatment orprevention of various diseases, including, for example, cancer,diabetes, neurodegenerative diseases, cardiovascular disease, bloodclotting, inflammation, flushing, obesity, aging, stress, etc. Invarious embodiments, combination therapies comprising asirtuin-modulating compound may refer to (1) pharmaceutical compositionsthat comprise one or more sirtuin-modulating compounds in combinationwith one or more therapeutic agents (e.g., one or more therapeuticagents described herein); and (2) co-administration of one or moresirtuin-modulating compounds with one or more therapeutic agents whereinthe sirtuin-modulating compound and therapeutic agent have not beenformulated in the same compositions (but may be present within the samekit or package, such as a blister pack or other multi-chamber package;connected, separately sealed containers (e.g., foil pouches) that can beseparated by the user; or a kit where the compound(s) and othertherapeutic agent(s) are in separate vessels). When using separateformulations, the sirtuin-modulating compound may be administeredsimultaneous with, intermittent with, staggered with, prior to,subsequent to, or combinations thereof, the administration of anothertherapeutic agent.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a compound described herein may alsocomprise increasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Increasing protein levels canbe achieved by introducing into a cell one or more copies of a nucleicacid that encodes a sirtuin. For example, the level of a sirtuin can beincreased in a mammalian cell by introducing into the mammalian cell anucleic acid encoding the sirtuin, e.g., increasing the level of SIRT1by introducing a nucleic acid encoding the amino acid sequence set forthin GenBank Accession No. NP_036370 and/or increasing the level of SIRT3by introducing a nucleic acid encoding the amino acid sequence set forthin GenBank Accession No. AAH01042.

A nucleic acid that is introduced into a cell to increase the proteinlevel of a sirtuin may encode a protein that is at least about 80%, 85%,90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, e.g.,SIRT1 and/or SIRT3 protein. For example, the nucleic acid encoding theprotein may be at least about 80%, 85%, 90%, 95%, 98%, or 99% identicalto a nucleic acid encoding a SIRT1 (e.g. GenBank Accession No.NM_012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042) protein.The nucleic acid may also be a nucleic acid that hybridizes, preferablyunder stringent hybridization conditions, to a nucleic acid encoding awild-type sirtuin, e.g., SIRT1 and/or SIRT3 protein. Stringenthybridization conditions may include hybridization and a wash in 0.2×SSCat 65° C. When using a nucleic acid that encodes a protein that isdifferent from a wild-type sirtuin protein, such as a protein that is afragment of a wild-type sirtuin, the protein is preferably biologicallyactive, e.g., is capable of deacetylation. It is only necessary toexpress in a cell a portion of the sirtuin that is biologically active.For example, a protein that differs from wild-type SIRT1 having GenBankAccession No. NP_036370, preferably contains the core structure thereof.The core structure sometimes refers to amino acids 62-293 of GenBankAccession No. NP_036370, which are encoded by nucleotides 237 to 932 ofGenBank Accession No. NM_012238, which encompasses the NAD binding aswell as the substrate binding domains. The core domain of SIRT1 may alsorefer to about amino acids 261 to 447 of GenBank Accession No.NP_036370, which are encoded by nucleotides 834 to 1394 of GenBankAccession No. NM_012238; to about amino acids 242 to 493 of GenBankAccession No. NP_036370, which are encoded by nucleotides 777 to 1532 ofGenBank Accession No. NM_012238; or to about amino acids 254 to 495 ofGenBank Accession No. NP_036370, which are encoded by nucleotides 813 to1538 of GenBank Accession No. NM_012238. Whether a protein retains abiological function, e.g., deacetylation capabilities, can be determinedaccording to methods known in the art.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise decreasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Decreasing a sirtuin proteinlevel can be achieved according to methods known in the art. Forexample, an siRNA, an antisense nucleic acid, or a ribozyme targeted tothe sirtuin can be expressed in the cell. A dominant negative sirtuinmutant, e.g., a mutant that is not capable of deacetylating, may also beused. For example, mutant H363Y of SIRT1, described, e.g., in Luo et al.(2001) Cell 107:137 can be used. Alternatively, agents that inhibittranscription can be used.

Methods for modulating sirtuin protein levels also include methods formodulating the transcription of genes encoding sirtuins, methods forstabilizing/destabilizing the corresponding mRNAs, and other methodsknown in the art.

Aging/Stress

In one aspect, the invention provides a method extending the lifespan ofa cell, extending the proliferative capacity of a cell, slowing aging ofa cell, promoting the survival of a cell, delaying cellular senescencein a cell, mimicking the effects of calorie restriction, increasing theresistance of a cell to stress, or preventing apoptosis of a cell, bycontacting the cell with a sirtuin-modulating compound of the inventionthat increases the level and/or activity of a sirtuin protein. In anexemplary embodiment, the methods comprise contacting the cell with asirtuin-modulating compound.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (i.e., cells obtained from anorganism, e.g., a human), may be kept alive in a cell culture. Embryonicstem (ES) cells and pluripotent cells, and cells differentiatedtherefrom, may also be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein to keep thecells, or progeny thereof, in culture for longer periods of time. Suchcells can also be used for transplantation into a subject, e.g., afterex vivo modification.

In one aspect, cells that are intended to be preserved for long periodsof time may be treated with a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein. The cells may be insuspension (e.g., blood cells, serum, biological growth media, etc.) orin tissues or organs. For example, blood collected from an individualfor purposes of transfusion may be treated with a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin proteinto preserve the blood cells for longer periods of time. Additionally,blood to be used for forensic purposes may also be preserved using asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein. Other cells that may be treated to extend theirlifespan or protect against apoptosis include cells for consumption,e.g., cells from non-human mammals (such as meat) or plant cells (suchas vegetables).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be applied during developmental and growthphases in mammals, plants, insects or microorganisms, in order to, e.g.,alter, retard or accelerate the developmental and/or growth process.

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to treat cells usefulfor transplantation or cell therapy, including, for example, solidtissue grafts, organ transplants, cell suspensions, stem cells, bonemarrow cells, etc. The cells or tissue may be an autograft, anallograft, a syngraft or a xenograft. The cells or tissue may be treatedwith the sirtuin-modulating compound prior toadministration/implantation, concurrently withadministration/implantation, and/or post administration/implantationinto a subject. The cells or tissue may be treated prior to removal ofthe cells from the donor individual, ex vivo after removal of the cellsor tissue from the donor individual, or post implantation into therecipient. For example, the donor or recipient individual may be treatedsystemically with a sirtuin-modulating compound or may have a subset ofcells/tissue treated locally with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. In certainembodiments, the cells or tissue (or donor/recipient individuals) mayadditionally be treated with another therapeutic agent useful forprolonging graft survival, such as, for example, an immunosuppressiveagent, a cytokine, an angiogenic factor, etc.

In yet other embodiments, cells may be treated with a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin proteinin vivo, e.g., to increase their lifespan or prevent apoptosis. Forexample, skin can be protected from aging (e.g., developing wrinkles,loss of elasticity, etc.) by treating skin or epithelial cells with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein. In an exemplary embodiment, skin is contacted with apharmaceutical or cosmetic composition comprising a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.Exemplary skin afflictions or skin conditions that may be treated inaccordance with the methods described herein include disorders ordiseases associated with or caused by inflammation, sun damage ornatural aging. For example, the compositions find utility in theprevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpemphigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, psoriasis, skin cancer and the effects of naturalaging. In another embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be used for thetreatment of wounds and/or burns to promote healing, including, forexample, first-, second- or third-degree burns and/or thermal, chemicalor electrical burns. The formulations may be administered topically, tothe skin or mucosal tissue.

Topical formulations comprising one or more sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused as preventive, e.g., chemopreventive, compositions. When used in achemopreventive method, susceptible skin is treated prior to any visiblecondition in a particular individual.

Sirtuin-modulating compounds may be delivered locally or systemically toa subject. In certain embodiments, a sirtuin-modulating compound isdelivered locally to a tissue or organ of a subject by injection,topical formulation, etc.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used for treating orpreventing a disease or condition induced or exacerbated by cellularsenescence in a subject; methods for decreasing the rate of senescenceof a subject, e.g., after onset of senescence; methods for extending thelifespan of a subject; methods for treating or preventing a disease orcondition relating to lifespan; methods for treating or preventing adisease or condition relating to the proliferative capacity of cells;and methods for treating or preventing a disease or condition resultingfrom cell damage or death. In certain embodiments, the method does notact by decreasing the rate of occurrence of diseases that shorten thelifespan of a subject. In certain embodiments, a method does not act byreducing the lethality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered to asubject in order to generally increase the lifespan of its cells and toprotect its cells against stress and/or against apoptosis. It isbelieved that treating a subject with a compound described herein issimilar to subjecting the subject to hormesis, i.e., mild stress that isbeneficial to organisms and may extend their lifespan.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to a subject to prevent aging andaging-related consequences or diseases, such as stroke, heart disease,heart failure, arthritis, high blood pressure, and Alzheimer's disease.Other conditions that can be treated include ocular disorders, e.g.,associated with the aging of the eye, such as cataracts, glaucoma, andmacular degeneration. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can also be administered tosubjects for treatment of diseases, e.g., chronic diseases, associatedwith cell death, in order to protect the cells from cell death.Exemplary diseases include those associated with neural cell death,neuronal dysfunction, or muscular cell death or dysfunction, such asParkinson's disease, Alzheimer's disease, multiple sclerosis,amyotrophic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasia such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; and graftrejections. Cell death can also be caused by surgery, drug therapy,chemical exposure or radiation exposure.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can also be administered to a subject suffering froman acute disease, e.g., damage to an organ or tissue, e.g., a subjectsuffering from stroke or myocardial infarction or a subject sufferingfrom a spinal cord injury. Sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may also be used torepair an alcoholic's liver.

Cardiovascular Disease

In another embodiment, the invention provides a method for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein include cardiomyopathy or myocarditis; such asidiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholiccardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy,and hypertensive cardiomyopathy. Also treatable or preventable usingcompounds and methods described herein are atheromatous disorders of themajor blood vessels (macrovascular disease) such as the aorta, thecoronary arteries, the carotid arteries, the cerebrovascular arteries,the renal arteries, the iliac arteries, the femoral arteries, and thepopliteal arteries. Other vascular diseases that can be treated orprevented include those related to platelet aggregation, the retinalarterioles, the glomerular arterioles, the vasa nervorum, cardiacarterioles, and associated capillary beds of the eye, the kidney, theheart, and the central and peripheral nervous systems. Thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used for increasing HDL levels in plasmaof an individual.

Yet other disorders that may be treated with sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteininclude restenosis, e.g., following coronary intervention, and disordersrelating to an abnormal level of high density and low densitycholesterol.

In certain embodiments, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapy with another cardiovascular agent. In certainembodiments, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapy with an anti-arrhythmia agent. In anotherembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapy with another cardiovascular agent.

Cell Death/Cancer

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to subjects who have recentlyreceived or are likely to receive a dose of radiation or toxin. Incertain embodiments, the dose of radiation or toxin is received as partof a work-related or medical procedure, e.g., administered as aprophylactic measure. In another embodiment, the radiation or toxinexposure is received unintentionally. In such a case, the compound ispreferably administered as soon as possible after the exposure toinhibit apoptosis and the subsequent development of acute radiationsyndrome.

Sirtuin-modulating compounds may also be used for treating and/orpreventing cancer. In certain embodiments, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedfor treating and/or preventing cancer. Calorie restriction has beenlinked to a reduction in the incidence of age-related disordersincluding cancer. Accordingly, an increase in the level and/or activityof a sirtuin protein may be useful for treating and/or preventing theincidence of age-related disorders, such as, for example, cancer.Exemplary cancers that may be treated using a sirtuin-modulatingcompound are those of the brain and kidney; hormone-dependent cancersincluding breast, prostate, testicular, and ovarian cancers; lymphomas,and leukemias. In cancers associated with solid tumors, a modulatingcompound may be administered directly into the tumor. Cancer of bloodcells, e.g., leukemia, can be treated by administering a modulatingcompound into the blood stream or into the bone marrow. Benign cellgrowth, e.g., warts, can also be treated. Other diseases that can betreated include autoimmune diseases, e.g., systemic lupus erythematosus,scleroderma, and arthritis, in which autoimmune cells should be removed.Viral infections such as herpes, HIV, adenovirus, and HTLV-1 associatedmalignant and benign disorders can also be treated by administration ofsirtuin-modulating compound. Alternatively, cells can be obtained from asubject, treated ex vivo to remove certain undesirable cells, e.g.,cancer cells, and administered back to the same or a different subject.

Chemotherapeutic agents may be co-administered with modulating compoundsdescribed herein as having anti-cancer activity, e.g., compounds thatinduce apoptosis, compounds that reduce lifespan or compounds thatrender cells sensitive to stress. Chemotherapeutic agents may be used bythemselves with a sirtuin-modulating compound described herein asinducing cell death or reducing lifespan or increasing sensitivity tostress and/or in combination with other chemotherapeutics agents. Inaddition to conventional chemotherapeutics, the sirtuin-modulatingcompounds described herein may also be used with antisense RNA, RNAi orother polynucleotides to inhibit the expression of the cellularcomponents that contribute to unwanted cellular proliferation.

Combination therapies comprising sirtuin-modulating compounds and aconventional chemotherapeutic agent may be advantageous over combinationtherapies known in the art because the combination allows theconventional chemotherapeutic agent to exert greater effect at lowerdosage. In a preferred embodiment, the effective dose (ED₅₀) for achemotherapeutic agent, or combination of conventional chemotherapeuticagents, when used in combination with a sirtuin-modulating compound isat least 2 fold less than the ED₅₀ for the chemotherapeutic agent alone,and even more preferably at 5 fold, 10 fold or even 25 fold less.Conversely, the therapeutic index (TI) for such chemotherapeutic agentor combination of such chemotherapeutic agent when used in combinationwith a sirtuin-modulating compound described herein can be at least 2fold greater than the TI for conventional chemotherapeutic regimenalone, and even more preferably at 5 fold, 10 fold or even 25 foldgreater.

Neuronal Diseases/Disorders

In certain aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat patientssuffering from neurodegenerative diseases, and traumatic or mechanicalinjury to the central nervous system (CNS), spinal cord or peripheralnervous system (PNS). Neurodegenerative disease typically involvesreductions in the mass and volume of the human brain, which may be dueto the atrophy and/or death of brain cells, which are far more profoundthan those in a healthy person that are attributable to aging.Neurodegenerative diseases can evolve gradually, after a long period ofnormal brain function, due to progressive degeneration (e.g., nerve celldysfunction and death) of specific brain regions. Alternatively,neurodegenerative diseases can have a quick onset, such as thoseassociated with trauma or toxins. The actual onset of brain degenerationmay precede clinical expression by many years. Examples ofneurodegenerative diseases include, but are not limited to, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewybody disease, chorea-acanthocytosis, primary lateral sclerosis, oculardiseases (ocular neuritis), chemotherapy-induced neuropathies (e.g.,from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathiesand Friedreich's ataxia. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can be used to treat thesedisorders and others as described below.

AD is a CNS disorder that results in memory loss, unusual behavior,personality changes, and a decline in thinking abilities. These lossesare related to the death of specific types of brain cells and thebreakdown of connections and their supporting network (e.g. glial cells)between them. The earliest symptoms include loss of recent memory,faulty judgment, and changes in personality. PD is a CNS disorder thatresults in uncontrolled body movements, rigidity, tremor, anddyskinesia, and is associated with the death of brain cells in an areaof the brain that produces dopamine. ALS (motor neuron disease) is a CNSdisorder that attacks the motor neurons, components of the CNS thatconnect the brain to the skeletal muscles.

HD is another neurodegenerative disease that causes uncontrolledmovements, loss of intellectual faculties, and emotional disturbance.Tay-Sachs disease and Sandhoff disease are glycolipid storage diseaseswhere GM2 ganglioside and related glycolipids substrates forβ-hexosaminidase accumulate in the nervous system and trigger acuteneurodegeneration.

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease, whichcan be treated with sirtuin-modulating compounds of the invention.

Neuronal loss is also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for treating orpreventing neuronal loss due to these prior diseases.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used to treat orprevent any disease or disorder involving axonopathy. Distal axonopathyis a type of peripheral neuropathy that results from some metabolic ortoxic derangement of peripheral nervous system (PNS) neurons. It is themost common response of nerves to metabolic or toxic disturbances, andas such may be caused by metabolic diseases such as diabetes, renalfailure, deficiency syndromes such as malnutrition and alcoholism, orthe effects of toxins or drugs. Those with distal axonopathies usuallypresent with symmetrical glove-stocking sensori-motor disturbances. Deeptendon reflexes and autonomic nervous system (ANS) functions are alsolost or diminished in affected areas.

Diabetic neuropathies are neuropathic disorders that are associated withdiabetes mellitus. Relatively common conditions which may be associatedwith diabetic neuropathy include third nerve palsy; mononeuropathy;mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy;autonomic neuropathy; and thoracoabdominal neuropathy.

Peripheral neuropathy is the medical term for damage to nerves of theperipheral nervous system, which may be caused either by diseases of thenerve or from the side-effects of systemic illness. Major causes ofperipheral neuropathy include seizures, nutritional deficiencies, andHIV, though diabetes is the most likely cause.

In an exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treat orprevent multiple sclerosis (MS), including relapsing MS andmonosymptomatic MS, and other demyelinating conditions, such as, forexample, chronic inflammatory demyelinating polyneuropathy (CIDP), orsymptoms associated therewith.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treattrauma to the nerves, including, trauma due to disease, injury(including surgical intervention), or environmental trauma (e.g.,neurotoxins, alcoholism, etc.).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be useful to prevent, treat, and alleviatesymptoms of various PNS disorders. The term “peripheral neuropathy”encompasses a wide range of disorders in which the nerves outside of thebrain and spinal cord—peripheral nerves—have been damaged. Peripheralneuropathy may also be referred to as peripheral neuritis, or if manynerves are involved, the terms polyneuropathy or polyneuritis may beused.

PNS diseases treatable with sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein include: diabetes,leprosy, Charcot-Marie-Tooth disease, Guillain-Barré syndrome andBrachial Plexus Neuropathies (diseases of the cervical and firstthoracic roots, nerve trunks, cords, and peripheral nerve components ofthe brachial plexus.

In another embodiment, a sirtuin-modulating compound may be used totreat or prevent a polyglutamine disease. Exemplary polyglutaminediseases include Spinobulbar muscular atrophy (Kennedy disease),Huntington's Disease (HD), Dentatorubral-pallidoluysian atrophy (HawRiver syndrome), Spinocerebellar ataxia type 1, Spinocerebellar ataxiatype 2, Spinocerebellar ataxia type 3 (Machado-Joseph disease),Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, andSpinocerebellar ataxia type 17.

In certain embodiments, the invention provides a method to treat acentral nervous system cell to prevent damage in response to a decreasein blood flow to the cell. Typically the severity of damage that may beprevented will depend in large part on the degree of reduction in bloodflow to the cell and the duration of the reduction. In certainembodiments, apoptotic or necrotic cell death may be prevented. In stilla further embodiment, ischemic-mediated damage, such as cytotoxic edemaor central nervous system tissue anoxemia, may be prevented. In eachembodiment, the central nervous system cell may be a spinal cell or abrain cell.

Another aspect encompasses administrating a sirtuin-modulating compoundto a subject to treat a central nervous system ischemic condition. Anumber of central nervous system ischemic conditions may be treated bythe sirtuin-modulating compounds described herein. In certainembodiments, the ischemic condition is a stroke that results in any typeof ischemic central nervous system damage, such as apoptotic or necroticcell death, cytotoxic edema or central nervous system tissue anoxia. Thestroke may impact any area of the brain or be caused by any etiologycommonly known to result in the occurrence of a stroke. In onealternative of this embodiment, the stroke is a brain stem stroke. Inanother alternative of this embodiment, the stroke is a cerebellarstroke. In still another embodiment, the stroke is an embolic stroke. Inyet another alternative, the stroke may be a hemorrhagic stroke. In afurther embodiment, the stroke is a thrombotic stroke.

In yet another aspect, a sirtuin-modulating compound may be administeredto reduce infarct size of the ischemic core following a central nervoussystem ischemic condition. Moreover, a sirtuin-modulating compound mayalso be beneficially administered to reduce the size of the ischemicpenumbra or transitional zone following a central nervous systemischemic condition.

In certain embodiments, a combination drug regimen may include drugs orcompounds for the treatment or prevention of neurodegenerative disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more anti-neurodegeneration agents.

Blood Coagulation Disorders

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or preventblood coagulation disorders (or hemostatic disorders). As usedinterchangeably herein, the terms “hemostasis”, “blood coagulation,” and“blood clotting” refer to the control of bleeding, including thephysiological properties of vasoconstriction and coagulation. Bloodcoagulation assists in maintaining the integrity of mammaliancirculation after injury, inflammation, disease, congenital defect,dysfunction or other disruption. Further, the formation of blood clotsdoes not only limit bleeding in case of an injury (hemostasis), but maylead to serious organ damage and death in the context of atheroscleroticdiseases by occlusion of an important artery or vein. Thrombosis is thusblood clot formation at the wrong time and place.

Accordingly, the present invention provides anticoagulation andantithrombotic treatments aiming at inhibiting the formation of bloodclots in order to prevent or treat blood coagulation disorders, such asmyocardial infarction, stroke, loss of a limb by peripheral arterydisease or pulmonary embolism.

As used interchangeably herein, “modulating or modulation of hemostasis”and “regulating or regulation of hemostasis” includes the induction(e.g., stimulation or increase) of hemostasis, as well as the inhibition(e.g., reduction or decrease) of hemostasis.

In one aspect, the invention provides a method for reducing orinhibiting hemostasis in a subject by administering a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.The compositions and methods disclosed herein are useful for thetreatment or prevention of thrombotic disorders. As used herein, theterm “thrombotic disorder” includes any disorder or conditioncharacterized by excessive or unwanted coagulation or hemostaticactivity, or a hypercoagulable state. Thrombotic disorders includediseases or disorders involving platelet adhesion and thrombusformation, and may manifest as an increased propensity to formthromboses, e.g., an increased number of thromboses, thrombosis at anearly age, a familial tendency towards thrombosis, and thrombosis atunusual sites.

In another embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of blood coagulation disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinand one or more anti-coagulation or anti-thrombosis agents.

Weight Control

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing weight gain or obesity in a subject. For example,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used, for example, to treat or preventhereditary obesity, dietary obesity, hormone related obesity, obesityrelated to the administration of medication, to reduce the weight of asubject, or to reduce or prevent weight gain in a subject. A subject inneed of such a treatment may be a subject who is obese, likely to becomeobese, overweight, or likely to become overweight. Subjects who arelikely to become obese or overweight can be identified, for example,based on family history, genetics, diet, activity level, medicationintake, or various combinations thereof.

In yet other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered tosubjects suffering from a variety of other diseases and conditions thatmay be treated or prevented by promoting weight loss in the subject.Such diseases include, for example, high blood pressure, hypertension,high blood cholesterol, dyslipidemia, type 2 diabetes, insulinresistance, glucose intolerance, hyperinsulinemia, coronary heartdisease, angina pectoris, congestive heart failure, stroke, gallstones,cholecystitis and cholelithiasis, gout, osteoarthritis, obstructivesleep apnea and respiratory problems, some types of cancer (such asendometrial, breast, prostate, and colon), complications of pregnancy,poor female reproductive health (such as menstrual irregularities,infertility, irregular ovulation), bladder control problems (such asstress incontinence); uric acid nephrolithiasis; psychological disorders(such as depression, eating disorders, distorted body image, and lowself-esteem). Finally, patients with AIDS can develop lipodystrophy orinsulin resistance in response to combination therapies for AIDS.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for inhibitingadipogenesis or fat cell differentiation, whether in vitro or in vivo.Such methods may be used for treating or preventing obesity.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for reducingappetite and/or increasing satiety, thereby causing weight loss oravoidance of weight gain. A subject in need of such a treatment may be asubject who is overweight, obese or a subject likely to becomeoverweight or obese. The method may comprise administering daily or,every other day, or once a week, a dose, e.g., in the form of a pill, toa subject. The dose may be an “appetite reducing dose.”

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing weight gain or obesity.For example, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-obesity agents.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered to reducedrug-induced weight gain. For example, a sirtuin-modulating compoundthat increases the level and/or activity of a sirtuin protein may beadministered as a combination therapy with medications that maystimulate appetite or cause weight gain, in particular, weight gain dueto factors other than water retention.

Metabolic Disorders/Diabetes

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing a metabolic disorder, such as insulin-resistance, apre-diabetic state, type II diabetes, and/or complications thereof.Administration of a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may increase insulin sensitivityand/or decrease insulin levels in a subject. A subject in need of such atreatment may be a subject who has insulin resistance or other precursorsymptom of type II diabetes, who has type II diabetes, or who is likelyto develop any of these conditions. For example, the subject may be asubject having insulin resistance, e.g., having high circulating levelsof insulin and/or associated conditions, such as hyperlipidemia,dyslipogenesis, hypercholesterolemia, impaired glucose tolerance, highblood glucose sugar level, other manifestations of syndrome X,hypertension, atherosclerosis and lipodystrophy.

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing a metabolic disorder. Forexample, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-diabetic agents.

Inflammatory Diseases

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or prevent adisease or disorder associated with inflammation. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered prior to the onset of, at, or after the initiationof inflammation. When used prophylactically, the compounds arepreferably provided in advance of any inflammatory response or symptom.Administration of the compounds may prevent or attenuate inflammatoryresponses or symptoms.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat orprevent allergies and respiratory conditions, including asthma,bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity,emphysema, chronic bronchitis, acute respiratory distress syndrome, andany chronic obstructive pulmonary disease (COPD). The compounds may beused to treat chronic hepatitis infection, including hepatitis B andhepatitis C.

Additionally, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to treat autoimmunediseases, and/or inflammation associated with autoimmune diseases, suchas arthritis, including rheumatoid arthritis, psoriatic arthritis, andankylosing spondylitis, as well as organ-tissue autoimmune diseases(e.g., Raynaud's syndrome), ulcerative colitis, Crohn's disease, oralmucositis, scleroderma, myasthenia gravis, transplant rejection,endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiplesclerosis, autoimmune thyroiditis, uveitis, systemic lupuserythematosis, Addison's disease, autoimmune polyglandular disease (alsoknown as autoimmune polyglandular syndrome), and Grave's disease.

In certain embodiments, one or more sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein may be takenalone or in combination with other compounds useful for treating orpreventing inflammation.

Flushing

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for reducing theincidence or severity of flushing and/or hot flashes which are symptomsof a disorder. For instance, the subject method includes the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein, alone or in combination with other agents, forreducing incidence or severity of flushing and/or hot flashes in cancerpatients. In other embodiments, the method provides for the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce the incidence or severity of flushing and/orhot flashes in menopausal and post-menopausal woman.

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used as a therapy forreducing the incidence or severity of flushing and/or hot flashes whichare side-effects of another drug therapy, e.g., drug-induced flushing.In certain embodiments, a method for treating and/or preventingdrug-induced flushing comprises administering to a patient in needthereof a formulation comprising at least one flushing inducing compoundand at least one sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. In other embodiments, a method fortreating drug induced flushing comprises separately administering one ormore compounds that induce flushing and one or more sirtuin-modulatingcompounds, e.g., wherein the sirtuin-modulating compound and flushinginducing agent have not been formulated in the same compositions. Whenusing separate formulations, the sirtuin-modulating compound may beadministered (1) at the same as administration of the flushing inducingagent, (2) intermittently with the flushing inducing agent, (3)staggered relative to administration of the flushing inducing agent, (4)prior to administration of the flushing inducing agent, (5) subsequentto administration of the flushing inducing agent, and (6) variouscombination thereof. Exemplary flushing inducing agents include, forexample, niacin, raloxifene, antidepressants, anti-psychotics,chemotherapeutics, calcium channel blockers, and antibiotics.

In certain embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of a vasodilator or an antilipemic agent(including anticholesteremic agents and lipotropic agents). In anexemplary embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used to reduceflushing associated with the administration of niacin.

In another embodiment, the invention provides a method for treatingand/or preventing hyperlipidemia with reduced flushing side effects. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of raloxifene. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of antidepressants oranti-psychotic agent. For instance, sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein can be used inconjunction (administered separately or together) with a serotoninreuptake inhibitor, or a 5HT2 receptor antagonist.

In certain embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used as part of atreatment with a serotonin reuptake inhibitor (SRI) to reduce flushing.In still another representative embodiment, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedto reduce flushing side effects of chemotherapeutic agents, such ascyclophosphamide and tamoxifen.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of antibiotics. For example, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteincan be used in combination with levofloxacin.

Ocular Disorders

One aspect of the present invention is a method for inhibiting, reducingor otherwise treating vision impairment by administering to a patient atherapeutic dosage of sirtuin modulator selected from a compounddisclosed herein, or a pharmaceutically acceptable salt, prodrug or ametabolic derivative thereof.

In certain aspects of the invention, the vision impairment is caused bydamage to the optic nerve or central nervous system. In particularembodiments, optic nerve damage is caused by high intraocular pressure,such as that created by glaucoma. In other particular embodiments, opticnerve damage is caused by swelling of the nerve, which is oftenassociated with an infection or an immune (e.g., autoimmune) responsesuch as in optic neuritis.

In certain aspects of the invention, the vision impairment is caused byretinal damage. In particular embodiments, retinal damage is caused bydisturbances in blood flow to the eye (e.g., arteriosclerosis,vasculitis). In particular embodiments, retinal damage is caused bydisruption of the macula (e.g., exudative or non-exudative maculardegeneration).

Exemplary retinal diseases include Exudative Age Related MacularDegeneration, Nonexudative Age Related Macular Degeneration, RetinalElectronic Prosthesis and RPE Transplantation Age Related MacularDegeneration, Acute Multifocal Placoid Pigment Epitheliopathy, AcuteRetinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, BranchRetinal Vein Occlusion, Cancer Associated and Related AutoimmuneRetinopathies, Central Retinal Artery Occlusion, Central Retinal VeinOcclusion, Central Serous Chorioretinopathy, Eales Disease, EpimacularMembrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema,Irvine-Gass Macular Edema, Macular Hole, Subretinal NeovascularMembranes, Diffuse Unilateral Subacute Neuroretinitis, NonpseudophakicCystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome,Exudative Retinal Detachment, Postoperative Retinal Detachment,Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment,Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis,Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy,Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy,Hemoglobinopathies Retinopathy, Purtscher Retinopathy, ValsalvaRetinopathy, Juvenile Retinoschisis, Senile Retinoschisis, TersonSyndrome and White Dot Syndromes.

Other exemplary diseases include ocular bacterial infections (e.g.conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viralinfections (e.g., Ocular Herpes Simplex Virus, Varicella Zoster Virus,Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as wellas progressive outer retinal necrosis secondary to HIV or otherHIV-associated and other immunodeficiency-associated ocular diseases. Inaddition, ocular diseases include fungal infections (e.g., Candidachoroiditis, histoplasmosis), protozoal infections (e.g., toxoplasmosis)and others such as ocular toxocariasis and sarcoidosis.

One aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing treatment with achemotherapeutic drug (e.g., a neurotoxic drug, or a drug that raisesintraocular pressure, such as a steroid), by administering to thesubject in need of such treatment a therapeutic dosage of a sirtuinmodulator disclosed herein.

Another aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing surgery, includingocular or other surgeries performed in the prone position such as spinalcord surgery, by administering to the subject in need of such treatmenta therapeutic dosage of a sirtuin modulator disclosed herein. Ocularsurgeries include cataract, iridotomy and lens replacements.

Another aspect of the invention is the treatment, including inhibitionand prophylactic treatment, of age related ocular diseases includecataracts, dry eye, age-related macular degeneration (AMD), retinaldamage and the like, by administering to the subject in need of suchtreatment a therapeutic dosage of a sirtuin modulator disclosed herein.

Another aspect of the invention is the prevention or treatment of damageto the eye caused by stress, chemical insult or radiation, byadministering to the subject in need of such treatment a therapeuticdosage of a sirtuin modulator disclosed herein. Radiation orelectromagnetic damage to the eye can include that caused by CRT's orexposure to sunlight or UV.

In certain embodiments, a combination drug regimen may include drugs orcompounds for the treatment or prevention of ocular disorders orsecondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more therapeutic agents for the treatment of an ocular disorder.

In certain embodiments, a sirtuin modulator can be administered inconjunction with a therapy for reducing intraocular pressure. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing glaucoma. In yet anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing optic neuritis. In certainembodiments, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing CMV Retinopathy. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing multiple sclerosis.

Mitochondrial-Associated Diseases and Disorders

In certain embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity. The methods involve administering to a subject in need thereofa therapeutically effective amount of a sirtuin-modulating compound.Increased mitochondrial activity refers to increasing activity of themitochondria while maintaining the overall numbers of mitochondria(e.g., mitochondrial mass), increasing the numbers of mitochondriathereby increasing mitochondrial activity (e.g., by stimulatingmitochondrial biogenesis), or combinations thereof. In certainembodiments, diseases and disorders that would benefit from increasedmitochondrial activity include diseases or disorders associated withmitochondrial dysfunction.

In certain embodiments, methods for treating diseases or disorders thatwould benefit from increased mitochondrial activity may compriseidentifying a subject suffering from a mitochondrial dysfunction.Methods for diagnosing a mitochondrial dysfunction may involve moleculargenetics, pathologic and/or biochemical analyses. Diseases and disordersassociated with mitochondrial dysfunction include diseases and disordersin which deficits in mitochondrial respiratory chain activity contributeto the development of pathophysiology of such diseases or disorders in amammal. Diseases or disorders that would benefit from increasedmitochondrial activity generally include for example, diseases in whichfree radical mediated oxidative injury leads to tissue degeneration,diseases in which cells inappropriately undergo apoptosis, and diseasesin which cells fail to undergo apoptosis.

In certain embodiments, the invention provides methods for treating adisease or disorder that would benefit from increased mitochondrialactivity that involves administering to a subject in need thereof one ormore sirtuin-modulating compounds in combination with anothertherapeutic agent such as, for example, an agent useful for treatingmitochondrial dysfunction or an agent useful for reducing a symptomassociated with a disease or disorder involving mitochondrialdysfunction.

In exemplary embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity by administering to a subject a therapeutically effectiveamount of a sirtuin-modulating compound. Exemplary diseases or disordersinclude, for example, neuromuscular disorders (e.g., Friedreich'sAtaxia, muscular dystrophy, multiple sclerosis, etc.), disorders ofneuronal instability (e.g., seizure disorders, migraine, etc.),developmental delay, neurodegenerative disorders (e.g., Alzheimer'sDisease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.),ischemia, renal tubular acidosis, age-related neurodegeneration andcognitive decline, chemotherapy fatigue, age-related orchemotherapy-induced menopause or irregularities of menstrual cycling orovulation, mitochondrial myopathies, mitochondrial damage (e.g., calciumaccumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), andmitochondrial deregulation.

Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction, such asDuchenne muscular dystrophy. In certain embodiments, sirtuin-modulatingcompounds may be used for reducing the rate of decline in muscularfunctional capacities and for improving muscular functional status inpatients with muscular dystrophy.

In certain embodiments, sirtuin-modulating compounds may be useful fortreatment mitochondrial myopathies. Mitochondrial myopathies range frommild, slowly progressive weakness of the extraocular muscles to severe,fatal infantile myopathies and multisystem encephalomyopathies. Somesyndromes have been defined, with some overlap between them. Establishedsyndromes affecting muscle include progressive external ophthalmoplegia,the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy,cardiac conduction defects, cerebellar ataxia, and sensorineuraldeafness), the MELAS syndrome (mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes), the MERFF syndrome (myoclonicepilepsy and ragged red fibers), limb-girdle distribution weakness, andinfantile myopathy (benign or severe and fatal).

In certain embodiments, sirtuin-modulating compounds may be useful fortreating patients suffering from toxic damage to mitochondria, such as,toxic damage due to calcium accumulation, excitotoxicity, nitric oxideexposure, drug induced toxic damage, or hypoxia.

In certain embodiments, sirtuin-modulating compounds may be useful fortreating diseases or disorders associated with mitochondrialderegulation.

Muscle Performance

In other embodiments, the invention provides methods for enhancingmuscle performance by administering a therapeutically effective amountof a sirtuin-modulating compound. For example, sirtuin-modulatingcompounds may be useful for improving physical endurance (e.g., abilityto perform a physical task such as exercise, physical labor, sportsactivities, etc.), inhibiting or retarding physical fatigues, enhancingblood oxygen levels, enhancing energy in healthy individuals, enhanceworking capacity and endurance, reducing muscle fatigue, reducingstress, enhancing cardiac and cardiovascular function, improving sexualability, increasing muscle ATP levels, and/or reducing lactic acid inblood. In certain embodiments, the methods involve administering anamount of a sirtuin-modulating compound that increase mitochondrialactivity, increase mitochondrial biogenesis, and/or increasemitochondrial mass.

Sports performance refers to the ability of the athlete's muscles toperform when participating in sports activities. Enhanced sportsperformance, strength, speed and endurance are measured by an increasein muscular contraction strength, increase in amplitude of musclecontraction, shortening of muscle reaction time between stimulation andcontraction. Athlete refers to an individual who participates in sportsat any level and who seeks to achieve an improved level of strength,speed and endurance in their performance, such as, for example, bodybuilders, bicyclists, long distance runners, short distance runners,etc. Enhanced sports performance in manifested by the ability toovercome muscle fatigue, ability to maintain activity for longer periodsof time, and have a more effective workout.

In the arena of athlete muscle performance, it is desirable to createconditions that permit competition or training at higher levels ofresistance for a prolonged period of time.

It is contemplated that the methods of the present invention will alsobe effective in the treatment of muscle related pathological conditions,including acute sarcopenia, for example, muscle atrophy and/or cachexiaassociated with burns, bed rest, limb immobilization, or major thoracic,abdominal, and/or orthopedic surgery.

In certain embodiments, the invention provides novel dietarycompositions comprising sirtuin modulators, a method for theirpreparation, and a method of using the compositions for improvement ofsports performance. Accordingly, provided are therapeutic compositions,foods and beverages that have actions of improving physical enduranceand/or inhibiting physical fatigues for those people involved inbroadly-defined exercises including sports requiring endurance andlabors requiring repeated muscle exertions. Such dietary compositionsmay additional comprise electrolytes, caffeine, vitamins, carbohydrates,etc.

Other Uses

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used for treating or preventing viralinfections (such as infections by influenza, herpes or papilloma virus)or as antifungal agents. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered as part of a combination drug therapy with anothertherapeutic agent for the treatment of viral diseases. In anotherembodiment, sirtuin-modulating compounds that increase the level and/oractivity of a sirtuin protein may be administered as part of acombination drug therapy with another anti-fungal agent.

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, modulating compounds may be administered to farmanimals to improve their ability to withstand farming conditions longer.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance, and resistance to apoptosis in plants. In certainembodiments, a compound is applied to plants, e.g., on a periodic basis,or to fungi. In another embodiment, plants are genetically modified toproduce a compound. In another embodiment, plants and fruits are treatedwith a compound prior to picking and shipping to increase resistance todamage during shipping. Plant seeds may also be contacted with compoundsdescribed herein, e.g., to preserve them.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for modulatinglifespan in yeast cells. Situations in which it may be desirable toextend the lifespan of yeast cells include any process in which yeast isused, e.g., the making of beer, yogurt, and bakery items, e.g., bread.Use of yeast having an extended lifespan can result in using less yeastor in having the yeast be active for longer periods of time. Yeast orother mammalian cells used for recombinantly producing proteins may alsobe treated as described herein.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance and resistance to apoptosis in insects. In this embodiment,compounds would be applied to useful insects, e.g., bees and otherinsects that are involved in pollination of plants. In a specificembodiment, a compound would be applied to bees involved in theproduction of honey. Generally, the methods described herein may beapplied to any organism, e.g., eukaryote, which may have commercialimportance. For example, they can be applied to fish (aquaculture) andbirds (e.g., chicken and fowl).

Higher doses of sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may also be used as a pesticide byinterfering with the regulation of silenced genes and the regulation ofapoptosis during development. In this embodiment, a compound may beapplied to plants using a method known in the art that ensures thecompound is bio-available to insect larvae, and not to plants.

At least in view of the link between reproduction and longevity,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can be applied to affect the reproduction of organismssuch as insects, animals and microorganisms.

4. Assays

Yet other methods contemplated herein include screening methods foridentifying compounds or agents that modulate sirtuins. An agent may bea nucleic acid, such as an aptamer. Assays may be conducted in a cellbased or cell free format. For example, an assay may comprise incubating(or contacting) a sirtuin with a test agent under conditions in which asirtuin can be modulated by an agent known to modulate the sirtuin, andmonitoring or determining the level of modulation of the sirtuin in thepresence of the test agent relative to the absence of the test agent.The level of modulation of a sirtuin can be determined by determiningits ability to deacetylate a substrate. Exemplary substrates areacetylated peptides which can be obtained from BIOMOL (Plymouth Meeting,Pa.). Preferred substrates include peptides of p53, such as thosecomprising an acetylated K382. A particularly preferred substrate is theFluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptideArg-His-Lys-Lys. Other substrates are peptides from human histones H3and H4 or an acetylated amino acid. Substrates may be fluorogenic. Thesirtuin may be SIRT1, Sir2, SIRT3, or a portion thereof. For example,recombinant SIRT1 can be obtained from BIOMOL. The reaction may beconducted for about 30 minutes and stopped, e.g., with nicotinamide. TheHDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOLResearch Laboratories) may be used to determine the level ofacetylation. Similar assays are described in Bitterman et al. (2002) J.Biol. Chem. 277:45099. The level of modulation of the sirtuin in anassay may be compared to the level of modulation of the sirtuin in thepresence of one or more (separately or simultaneously) compoundsdescribed herein, which may serve as positive or negative controls.Sirtuins for use in the assays may be full length sirtuin proteins orportions thereof. Since it has been shown herein that activatingcompounds appear to interact with the N-terminus of SIRT1, proteins foruse in the assays include N-terminal portions of sirtuins, e.g., aboutamino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 ofSir2.

In certain embodiments, a screening assay comprises (i) contacting asirtuin with a test agent and an acetylated substrate under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test agent; and (ii) determining the level of acetylation of thesubstrate, wherein a lower level of acetylation of the substrate in thepresence of the test agent relative to the absence of the test agentindicates that the test agent stimulates deacetylation by the sirtuin,whereas a higher level of acetylation of the substrate in the presenceof the test agent relative to the absence of the test agent indicatesthat the test agent inhibits deacetylation by the sirtuin.

In another embodiment, the screening assay may detect the formation of a2′/3′-O-acetyl-ADP-ribose product of sirtuin-mediated NAD-dependentdeacetylation. This 0-acetyl-ADP-ribose product is formed in equimolarquantities with the deacetylated peptide product of the sirtuindeacetylation reaction. Accordingly, the screening assay may include (i)contacting a sirtuin with a test agent and an acetylated substrate underconditions appropriate for the sirtuin to deacetylate the substrate inthe absence of the test agent; and (ii) determining the amount ofO-acetyl-ADP-ribose formation, wherein an increase inO-acetyl-ADP-ribose formation in the presence of the test agent relativeto the absence of the test agent indicates that the test agentstimulates deacetylation by the sirtuin, while a decrease inO-acetyl-ADP-ribose formation in the presence of the test agent relativeto the absence of the test agent indicates that the test agent inhibitsdeacetylation by the sirtuin.

Methods for identifying an agent that modulates, e.g., stimulates,sirtuins in vivo may comprise (i) contacting a cell with a test agentand a substrate that is capable of entering a cell in the presence of aninhibitor of class I and class II HDACs under conditions appropriate forthe sirtuin to deacetylate the substrate in the absence of the testagent; and (ii) determining the level of acetylation of the substrate,wherein a lower level of acetylation of the substrate in the presence ofthe test agent relative to the absence of the test agent indicates thatthe test agent stimulates deacetylation by the sirtuin, whereas a higherlevel of acetylation of the substrate in the presence of the test agentrelative to the absence of the test agent indicates that the test agentinhibits deacetylation by the sirtuin. A preferred substrate is anacetylated peptide, which is also preferably fluorogenic, as furtherdescribed herein. The method may further comprise lysing the cells todetermine the level of acetylation of the substrate. Substrates may beadded to cells at a concentration ranging from about 1 μM to about 10mM, preferably from about 10 μM to 1 mM, even more preferably from about100 μM to 1 mM, such as about 200 μM. A preferred substrate is anacetylated lysine, e.g., ε-acetyl lysine (Fluor de Lys, FdL) or Fluor deLys-SIRT1. A preferred inhibitor of class I and class II HDACs istrichostatin A (TSA), which may be used at concentrations ranging fromabout 0.01 to 100 μM, preferably from about 0.1 to 10 μM, such as 1 μM.Incubation of cells with the test compound and the substrate may beconducted for about 10 minutes to 5 hours, preferably for about 1-3hours. Since TSA inhibits all class I and class II HDACs, and thatcertain substrates, e.g., Fluor de Lys, is a poor substrate for SIRT2and even less a substrate for SIRT3-7, such an assay may be used toidentify modulators of SIRT1 in vivo.

5. Pharmaceutical Compositions

The compounds described herein may be formulated in a conventionalmanner using one or more physiologically or pharmaceutically acceptablecarriers or excipients. For example, compounds and theirpharmaceutically acceptable salts and solvates may be formulated foradministration by, for example, injection (e.g. SubQ, IM, IP),inhalation or insufflation (either through the mouth or the nose) ororal, buccal, sublingual, transdermal, nasal, parenteral or rectaladministration. In certain embodiments, a compound may be administeredlocally, at the site where the target cells are present, i.e., in aspecific tissue, organ, or fluid (e.g., blood, cerebrospinal fluid,etc.).

The compounds can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For parenteral administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozenges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery), thecompounds may be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, compounds may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example,compounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Controlled release formula also includespatches.

In certain embodiments, the compounds described herein can be formulatedfor delivery to the central nervous system (CNS) (reviewed in Begley,Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approachesfor drug delivery to the CNS include: neurosurgical strategies (e.g.,intracerebral injection or intracerebroventricular infusion); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the BBB) in an attempt to exploit one ofthe endogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of an agent (e.g., conjugationof water-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide).

Liposomes are a further drug delivery system which is easily injectable.Accordingly, in the method of invention the active compounds can also beadministered in the form of a liposome delivery system. Liposomes arewell known by those skilled in the art. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine ofphosphatidylcholines. Liposomes usable for the method of inventionencompass all types of liposomes including, but not limited to, smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles.

Another way to produce a formulation, particularly a solution, of acompound described herein, is through the use of cyclodextrin. Bycyclodextrin is meant α-, β-, or γ-cyclodextrin. Cyclodextrins aredescribed in detail in Pitha et al., U.S. Pat. No. 4,727,064, which isincorporated herein by reference. Cyclodextrins are cyclic oligomers ofglucose; these compounds form inclusion complexes with any drug whosemolecule can fit into the lipophile-seeking cavities of the cyclodextrinmolecule.

Rapidly disintegrating or dissolving dosage forms are useful for therapid absorption, particularly buccal and sublingual absorption, ofpharmaceutically active agents. Fast melt dosage forms are beneficial topatients, such as aged and pediatric patients, who have difficulty inswallowing typical solid dosage forms, such as caplets and tablets.Additionally, fast melt dosage forms circumvent drawbacks associatedwith, for example, chewable dosage forms, wherein the length of time anactive agent remains in a patient's mouth plays an important role indetermining the amount of taste masking and the extent to which apatient may object to throat grittiness of the active agent.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more compounds described herein. In otherembodiments, the pharmaceutical composition comprises: (i) 0.05 to 1000mg of the compounds of the invention, or a pharmaceutically acceptablesalt thereof, and (ii) 0.1 to 2 grams of one or more pharmaceuticallyacceptable excipients.

In some embodiments, a compound described herein is incorporated into atopical formulation containing a topical carrier that is generallysuited to topical drug administration and comprising any such materialknown in the art. The topical carrier may be selected so as to providethe composition in the desired form, e.g., as an ointment, lotion,cream, microemulsion, gel, oil, solution, or the like, and may becomprised of a material of either naturally occurring or syntheticorigin. It is preferable that the selected carrier not adversely affectthe active agent or other components of the topical formulation.Examples of suitable topical carriers for use herein include water,alcohols and other nontoxic organic solvents, glycerin, mineral oil,silicone, petroleum jelly, lanolin, fatty acids, vegetable oils,parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams,microemulsions and gels.

The compounds may be incorporated into ointments, which generally aresemisolid preparations which are typically based on petrolatum or otherpetroleum derivatives. The specific ointment base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery, and, preferably, will provide for other desiredcharacteristics as well, e.g., emolliency or the like. As with othercarriers or vehicles, an ointment base should be inert, stable,nonirritating and nonsensitizing.

The compounds may be incorporated into lotions, which generally arepreparations to be applied to the skin surface without friction, and aretypically liquid or semiliquid preparations in which solid particles,including the active agent, are present in a water or alcohol base.Lotions are usually suspensions of solids, and may comprise a liquidoily emulsion of the oil-in-water type.

The compounds may be incorporated into creams, which generally areviscous liquid or semisolid emulsions, either oil-in-water orwater-in-oil. Cream bases are water-washable, and contain an oil phase,an emulsifier and an aqueous phase. The oil phase is generally comprisedof petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation, as explained in Remington's, supra, is generally anonionic, anionic, cationic or amphoteric surfactant.

The compounds may be incorporated into microemulsions, which generallyare thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids, such as oil and water, stabilized by an interfacialfilm of surfactant molecules (Encyclopedia of Pharmaceutical Technology(New York: Marcel Dekker, 1992), volume 9).

The compounds may be incorporated into gel formulations, which generallyare semisolid systems consisting of either suspensions made up of smallinorganic particles (two-phase systems) or large organic moleculesdistributed substantially uniformly throughout a carrier liquid (singlephase gels). Although gels commonly employ aqueous carrier liquid,alcohols and oils can be used as the carrier liquid as well.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In certain topical formulations, the active agent is present in anamount in the range of approximately 0.25 wt. % to 75 wt. % of theformulation, preferably in the range of approximately 0.25 wt. % to 30wt. % of the formulation, more preferably in the range of approximately0.5 wt. % to 15 wt. % of the formulation, and most preferably in therange of approximately 1.0 wt. % to 10 wt. % of the formulation.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a compound, or by insertion of asustained release device that releases a compound. A compound may bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye, as for example the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/ciliary, lens, choroid/retina and sclera. The pharmaceuticallyacceptable ophthalmic vehicle may, for example, be an ointment,vegetable oil or an encapsulating material. Alternatively, the compoundsof the invention may be injected directly into the vitreous and aqueoushumour. In a further alternative, the compounds may be administeredsystemically, such as by intravenous infusion or injection, fortreatment of the eye.

The compounds described herein may be stored in oxygen free environment.For example, a composition can be prepared in an airtight capsule fororal administration, such as Capsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a compound as described herein, can beadministered according to methods for administering a graft to asubject, which may be accompanied, e.g., by administration of animmunosuppressant drug, e.g., cyclosporin A. For general principles inmedicinal formulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996; and HematopoieticStem Cell Therapy, E. D. Ball, J. Lister & P. Law, ChurchillLivingstone, 2000.

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals. The LD₅₀ is the dose lethal to 50% of the population. The ED₅₀is the dose therapeutically effective in 50% of the population. The doseratio between toxic and therapeutic effects (LD₅₀/ED₅₀) is thetherapeutic index. Compounds that exhibit large therapeutic indexes arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for modulating the lifespan of cells or modulating apoptosis. A kitmay comprise one or more compounds as described herein, e.g., inpremeasured doses. A kit may optionally comprise devices for contactingcells with the compounds and instructions for use. Devices includesyringes, stents and other devices for introducing a compound into asubject (e.g., the blood vessel of a subject) or applying it to the skinof a subject.

In yet another embodiment, the invention provides a composition ofmatter comprising a compound of this invention and another therapeuticagent (the same ones used in combination therapies and combinationcompositions) in separate dosage forms, but associated with one another.The term “associated with one another” as used herein means that theseparate dosage forms are packaged together or otherwise attached to oneanother such that it is readily apparent that the separate dosage formsare intended to be sold and administered as part of the same regimen.The compound and the other agent are preferably packaged together in ablister pack or other multi-chamber package, or as connected, separatelysealed containers (such as foil pouches or the like) that can beseparated by the user (e.g., by tearing on score lines between the twocontainers).

In still another embodiment, the invention provides a kit comprising inseparate vessels, a) a compound of this invention; and b) anothertherapeutic agent such as those described elsewhere in thespecification.

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1. Preparation of(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-(2H)-carboxamideStep 1. Synthesis of (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)succinate

To a 2 L flask equipped with a thermometer, a reflux condenser, and amechanical stirrer was added (100 g, 0.52 mol) of2,6-dichloro-3-nitropyridine (205 g, 1.04 mol) of (S)-aspartic aciddimethyl ester hydrochloride (174 g, 2.07 mol) of NaHCO₃ and 1 L oftetrahydrofuran. The reaction was stirred at 40° C. for 16 h, and wasmonitored for the disappearance of 2,6-dichloropyridine by HPLC. Afterthe reaction was complete, the solids were filtered away and washed withethyl acetate (3×300 mL). The combined filtrate and washings wereconcentrated to dryness, and the residue was taken up in 1 L of ethylacetate. The solution was stirred with 200 g of charcoal at ambienttemperature for 2 h, and the charcoal was filtered away and washed withadditional ethyl acetate (3×200 mL). The combined filtrate and washingswere concentrated in vacuo to give the crude product (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)succinate (180 g) as a yellowoil. This was used in the next step without further purification. MS(ESI) calcd for C₁₁H₁₂ClN₃O₆: 317.0. found: 318.0 (M+H)⁺.

An analagous procedure could be used to prepare (R)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)succinate by starting with(R)-aspartic acid dimethyl ester hydrochloride.

Step 2. Synthesis of (S)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate

A 5 L three necked flask equipped with a thermometer, a refluxcondenser, and a mechanical stirrer was charged with crude (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)succinate (180 g, 0.52 mol) ofiron powder (146 g, 2.59 mol), 2 L of 2-propanol, and 700 mL of water.The mixture was stirred at 40° C., acetic acid (15.5 g, 0.259 mmol) wasadded at a rate sufficient to keep the inner temperature below 70° C.The reaction was stirred at 70° C. for 30 min, HPLC indicated that thereaction was complete. The mixture was cooled to 40° C., then Na₂CO₃(165 g, 1.55 mol) was added, and the mixture was stirred for 1 h. Thesolids were filtered away, then the solids were washed withtetrahydrofuran (3×500 mL). The combined filtrate and washings wereconcentrated in vacuo, then the residue was stirred in 1 L of ethanolfor 12 hrs. The solid was filtered and washed with cold ethanol. Thiswas dried in vacuo to give (S)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate(91 g, 68%) as an off-white solid. MS (ESI) calcd for C₁₀H₁₀ClN₃O₃:255.0. found: 256.0 (M+H)⁺.

An analogous procedure could be used to prepare (R)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate bystarting with (R)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)succinate.

Step 3. Synthesis of(S)-2-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol

A 5 L 3-necked flask equipped with a mechanical stirrer, a refluxcondenser, and a nitrogen inlet was charged with of LiAlH₄ (60 g, 1.58mol). The flask was cooled with an ice bath, then 500 mL oftetrahydrofuran was added. The stirred mixture was cooled to 0° C., asolution of (S)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate(81 g, 0.32 mol) in 2 L of tetrahydrofuran was added, while keeping theinternal temperature below 5° C. After the addition was complete, thereaction was heated at reflux for 16 h, monitoring by HPLC for theappearance of product. The ester reduction occurred rapidly, while thelactam reduction required longer for complete reduction. The reactionwas cooled to 5° C., then 60 mL of water was added, keeping the internaltemperature below 10° C. After addition was complete, the reaction wasstirred for 15 min. Next, 60 mL of 15% (w/w) NaOH_((aq)) was added,keeping the internal temperature below 5° C. After addition wascomplete, the reaction was stirred for 15 min. To complete the workup,180 mL of water was added, then the mixture was stirred at ambienttemperature for 1 h. The solids were filtered and washed withtetrahydrofuran (3×150 mL). The filtrate and washings were concentratedin vacuo, then the solid residue was dried in vacuo to give(S)-2-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol (55g, 81%) as a brown solid. MS (ESI) calcd for C₉H₁₂ClN₃O: 213.1. found:214.1 (M+H)⁺.

An analogous procedure could be used to prepare(R)-2-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol bystarting with (R)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate.

Step 4. Synthesis of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To a solution of(S)-2-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol (50g, 0.234 mol) in 500 mL of CH₂Cl₂ was added triethylamine (95 g, 0.936mol). The mixture was stirred at ambient temperature until it washomogeneous, then it was cooled to 0° C. Next, POCl₃ (54 g, 0.351 mol)was added, dropwise, maintaining the temperature at 0° C. 5. Cooling wasremoved, and the reaction was stirred at ambient temperature for 2 h andmonitored by HPLC for the disappearance of the starting alcohol. Afterthe reaction was complete, 200 mL of 1.2M NaNCO_(3(aq)) was added. Thelayers were separated, and the aqueous layer was extracted with CH₂Cl₂.The combined CH₂Cl₂ layers were extracted with 1M HCl_((aq)) (4×300 mL),and the combined HCl layers were adjusted to pH=8 with NaHCO_(3(sat.)).The resulting mixture was extracted with CH₂Cl₂ (4×300 mL), and thecombined CH₂Cl₂ layers were dried over Na₂SO₄, filtered, and treatedwith 50 g of charcoal. The mixture was stirred at ambient temperaturefor 3 h, filtered through charcoal, and the charcoal washed with anadditional 200 mL of CH₂Cl₂. The combined filtrate and wash solution wasconcentrated to dryness. The solid residue was dried in vacuo to give(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(30 g, 66%) as an off-white crystalline solid. MS (ESI) calcd forC₉H₁₀ClN₃: 195.1. found: 196.1 (M+H)⁺.

An analogous procedure could be used to prepare(4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineby starting with(R)-2-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol.

Step 5. Synthesis of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(1.88 g, 9.6 mmol), (3-trifluoromethylphenyl)boronic acid (2.4 g, 12.6mmol), Pd(OAc)₂ (0.228 g, 1.02 mmol),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (0.972 g, 2.04mmol), and Cs₂CO₃ (6.6 g, 20.4 mmol) was dissolved in dioxane/H₂O (50mL, v/v=9:1). The reaction mixture was heated to 90° C. overnight. Aftercooling to room temp., it was diluted with EtOAc (120 mL) and washedwith water. The aqueous layer was extracted with EtOAc, and the combinedorganic layers were washed with brine, dried over MgSO₄, filtered, andconcentrated. Purification by silica gel chromatography (0 to 100% EtOAcin pentane gradient) afforded(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineas a light yellow solid (2.26 g, 77%). MS (ESI) calcd for C₁₆H₁₄F₃N₃:305.1. found: 306 [M+H].

An analogous coupling procedure using Pd(OAc)₂ could be used to prepare(4S)-7-(3-substitutedphenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepines byusing the appropriate 3-substituted phenylboronic acids or esters. Theanalogous enantiomers could be made starting with(4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine.

Step 6. Synthesis of(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(0.100 g, 0.328 mmol) and Et₃N (160 μL, 1.15 mmol) in THF (4 mL) wasadded triphosgene (0.050 g, 0.164 mmol). After stirring for 30 min. atroom temp., 2-pyridylamine (0.092 g, 0.983 mmol) was added. The reactionmixture was heated to 60° C. overnight, and the reaction mixture wasconcentrated and the residue taken up in CH₂Cl₂ (30 mL). The solutionwas washed with water and brine, dried over MgSO₄, filtered, andconcentrated. Purification by silica gel chromatography (0 to 100% EtOAcin pentane gradient) afforded(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.086 g, 62%). MS (ESI) calcd for C₂₂H₁₈F₃N₅O: 425.2. found: 426.2[M+H].

An analogous procedure could be used to prepare a variety of(4S)-7-(3-trifluoromethylphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate amine moiety for 2-pyridylamine. Theanalogous enantiomers could be made by starting with(4R)-7-(3-trifluoromethylphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.

Analogously,(9S)-2-(3-(trifluoromethyl)phenyl)-N-(aryl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidescould be prepared by this general procedure from(9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocineand the appropriate amine moiety.

Example 2. Preparation of(4S)—N-phenethyl-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(0.100 g, 0.326 mmol) in CH₂Cl₂ (10 mL) was added pyridine (0.0775 g,0.980 mmol) and phenyl chloroformate (0.06117 g, 0.392 mmol) at 0° C.After 2 h, the mixture was quenched with sat. aqueous Na₂CO₃, extractedwith CH₂Cl₂ (3×75 mL), washed with brine, dried with Na₂SO₃, andconcentrated. The residue was purified by flash silica gelchromatography to give (4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(0.120 g, yield 84%).

(4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate,2-phenylethanamine (0.057 g, 0.470 mmol), and DMAP (0.035 g, 0.282 mmol)in MeCN (3 mL) were stirred at 80° C. overnight. After cooling to roomtemperature, the mixture was concentrated and the residue purified byPrep-TLC eluting with CH₂Cl₂:MeOH (10:1) to give(4S)—N-phenethyl-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.020 g, yield 18%). MS (ESI) calcd for C₂₅H₂₃F₃N₄O: 452.18. found: 453[M+H].

Example 3. Preparation of(4S)—N-(3-(3-aminoprop-1-yn-1-yl)-5-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A suspension of(4S)—N-(3-(3-aminoprop-1-yn-1-yl)-5-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(A, 0.050 g, 0.08 mmol) in 4N HCl indioxane (10 mL) was stirred for 16 hat room temp. The mixture was concentrated under reduced pressure andtriturated with CH₃CN. The residue was dissolved in CH₃CN:H₂O andlyophilized to give(4S)—N-(3-(3-aminoprop-1-yn-1-yl)-5-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.030 g, yield 70%). MS (ESI) calcd for C₂₉H₂₃F₃N₆O₂: 544.18. found:545 [M+H].

Example 4. Preparation of(4S)—N-methyl-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.267 g, 0.63 mmol) was dissolved in dimethylacetamide and oneequivalent of NaH (0.025 g, 60% in oil) was added. The solution wasstirred for 5 minutes before the addition of MeI (39 μL). The mixturewas stirred overnight at room temperature then diluted with ethylacetate and washed sequentially with brine (2×), water (2×), and brine.The solution was dried (Na₂SO₄) and concentrated under reduced pressureand loaded onto a silica gel cartridge (ethyl acetate: pentane eluent).Concentration of the pure fractions afforded pure(4S)—N-methyl-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.MS (ESI) calcd for C₂₃H₂₀F₃N₅O: 439.16. found: 440.1 [M+H].

Example 5. Preparation of(4S)—N-(3-fluoropyridin-4-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Phenyl chloroformate (1.46 g, 9.37 mmol) was added dropwise to a cooledsolution of 3-fluoropyridin-4-amine (1 g, 8.92 mmol) and pyridine (0.95mL, 11.16 mmol) in THF (10 mL). The reaction was stirred at room temp.overnight. Purification by prep-TLC afforded phenyl(3-fluoropyridin-4-yl)carbamate as a yellow solid.

A mixture of phenyl (3-fluoropyridin-4-yl)carbamate (72 mg, 0.31 mmol),(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(50 mg, 0.16 mmol) and DMAP (24 mg, 0.20 mmol) in 3 mL of acetonitrilewas stirred at 60° C. overnight. The mixture was directly loaded ontoprep-TLC and purified using ethyl acetate/pet.ether=1:3˜1:8 as eluent toafford(4S)—N-(3-fluoropyridin-4-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(18 mg, 25%) as a white solid. MS (ESI) calcd for C₂₂H₁₇F₄N₅O: 443.1.found: 444.1 [M+H].

This general urea formation procedure using phenyl carbamates could beused to prepare a variety of(4S)-7-(aryl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate(4S)-7-(aryl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepinefor(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineand by substituting the appropriate amine moiety for3-fluoropyridin-4-amine. The enantiomer series could be made by startingwith(4R)-7-(aryl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepines.

Example 6. Preparation of(4S)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To dioxane/H₂O (10 mL/1 mL) was added(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(500 mg, 2.56 mmol), (3-chlorophenyl)boronic acid (807 mg, 5.11 mmol),Pd(dppf)Cl₂ (212 mg, 0.26 mmol), and Cs₂CO₃ (2.08 g, 6.4 mmol). Themixture was stirred at 90° C. overnight. The mixture was concentratedand purified by column chromatography (ethyl acetate/pet. ether=1/4) toafford(4S)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(506 mg, 81%). MS (ESI) calcd for C₁₅H₁₄ClN₃: 271.1. found: 272.1 [M+H].

An analogous procedure could be used to prepare(4S)-7-(3-fluorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineor(4S)-7-(3-methoxyphenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineby using the appropriate boronic acid or ester. The enantiomer seriescould be made by starting with the appropriate (4R)-7-(3-substitutedphenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine. Alsomade via this method using the appropriate starting chloride were:(9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,(9R)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,3-((9R)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)benzonitrile,3-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)benzonitrile,(9S)-2-(5-(methylsulfonyl)pyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,(9S)-2-(5-(trifluoromethyl)pyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,N,N-dimethyl-3-((9R)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)aniline,N,N-dimethyl-3-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)aniline,(9S)-2-(6-methylpyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,(9S)-2-(pyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine,(9S)-2-(3-chlorophenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine.

Step 2. Synthesis of(4S)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Phenyl chloroformate (6.99 mL, 55.79 mmol) was added dropwise to acooled solution of 2-pyridylamine (5 g, 53.10 mmol) and pyridine (5.65mL, 66.41 mmol) in THF (50 mL). The reaction was stirred at room temp.overnight. Brine was added slowly and the mixture was extracted withethyl acetate. The layers were separated and organic layer was washedwith sat. sodium bicarbonate and brine. The organic layer was then driedover anhydrous sodium sulfate and concentrated under reduced pressure.The residue was washed with pet. ether to afford phenylpyridin-2-ylcarbamate (2.1 g, 18%).

A mixture of phenyl pyridin-2-ylcarbamate (66 mg, 0.30 mmol),(4S)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(40 mg, 0.15 mmol) and DMAP (23 mg, 0.18 mmol) in 3 mL of acetonitrilewas stirred at 65° C. overnight. Reaction progress was monitored by TLCand LC-MS. The mixture was directly loaded on prep-TLC using ethylacetate/petroleum ether=1:3˜1:8 as eluent to give(4S)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(28 mg, 48%) as a white solid. MS (ESI) calcd for C₂₁H₁₈ClN₅O: 391.1.found: 392.1 [M+H].

This general urea formation procedure using phenyl carbamates could beused to prepare a variety of (4S)-7-(3-chloro, -fluoro, or-methoxyphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using (4S)-7-(3-chloro, -fluoro or-methoxyphenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineand the appropriate amine moiety. The enantiomer series could be made bystarting with the appropriate (4R)-7-(3-substitutedphenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine.Analogously, (9S)-2-(5-fluoro- orchloropyridin-3-yl)-N-(aryl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidescould be prepared via this urea formation procedure; see the followingexample for preparation of the starting (9S)-2-(5-fluoro- orchloropyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocines.

The analogous procedure used to prepare(4S)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(steps 1 and 2 above) can be used to make the following compoundsstarting with commercially available boronic esters:

Example 7. Preparation of(9S)-2-(5-chloropyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocineStep 1. Synthesis of (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)glutarate

This moiety was made using the following protocol. To a mixture of 40.0g (207 mmol) of 2,6-dichloro-3-nitropyridine, 87.7 g (414 mmol) ofL-glutamic acid dimethyl ester hydrochloride, and 69.6 g (829 mmol) ofNaHCO₃ was added 600 mL of tetrahydrofuran. The mixture was stirred at40° C. for 24 h, monitoring for the disappearance of2,6-dichloro-3-nitropyridine by HPLC. After the reaction was complete,the solids were filtered away and washed with ethyl acetate (3×100 mL).The combined filtrate and washings were concentrated in vacuo, then theresidue was purified via silica gel chromatography, eluting with 10/1(v/v) hexanes/ethyl acetate to give 60 g (87%) of the product as ayellow solid. MS (ESI) calcd for C₁₂H₁₄ClN₃O₆: 331.0. found: 332.1(M+H)⁺.

Step 2. Synthesis of (S)-methyl3-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propanoate

This moiety was made using the following protocol. To a mixture of 20 g(60.2 mmol) of (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)pentanedioate, and 16.8 g (301mmol) of iron powder was added 375 mL of 2-propanol, then 125 mL ofwater. To the stirred mixture was added 5.5 g (90.3 mmol) of aceticacid, then the reaction was stirred at reflux for 1 h. The reaction wasmonitored for the disappearance of starting material by HPLC. After thereaction was complete, the solids were filtered off and washed with2-propanol (3×50 mL). The combined filtrate and washings wereconcentrated to dryness, then the residue was dried in vacuo to give 15g (81%) of the product as a dark yellow solid. This was used withoutfurther purification in the next step. MS (ESI) calcd for C₁₁H₁₂ClN₃O₃:269.0. found: 270.1.

Step 3. Synthesis of(S)-3-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propan-1-ol

This moiety was made using the following protocol. To a solution of17.78 g (133.3 mmol) of AlCl₃ in 260 mL of tetrahydrofuran (THF) underN₂ was added 200 mL of 2M LiAlH₄ in THF, dropwise, at a rate to controlgas evolution. This gave a solution of alane (AlH₃) in THF. In aseparate flask, a solution of 26.0 g (96.4 mmol) of (S)-methyl3-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propanoatein 460 mL of THF was prepared under N₂, then cooled with a dryice/acetone bath. To this was added the alane solution, dropwise withstirring, over 2 h. When the addition was complete, the cooling bath wasremoved, and the reaction was allowed to warm to ambient temperature.After 1.5 h, LCMS analysis showed that the reaction was complete. Next,a solution of 17.6 g NaOH in 65 mL of water was added slowly to controlthe evolution of H₂. The suspension was allowed to stir for 18 h, thenthe solids were filtered away. The precipitate was washed with ethylacetate, then the filtrate and washings were concentrated in vacuo. Theproduct was purified via silica gel chromatography, (330 g prepackedcolumn) eluting with CH₂Cl₂, followed by a gradient of 0 to 10% methanolin CH₂Cl₂ to give 15.21 g (69%) of a yellow-orange solid. MS (ESI) calcdfor C₁₀H₁₄ClN₃O: 227.1. found: 228.1.

Step 4. Synthesis of(5R,9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

To 12 g (52.7 mmol) of(S)-3-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propan-1-olwas added 160 mL of 48% (w/w) HBr_((aq)), then the reaction was stirredat 90° C. for 18 h. The reaction was monitored by HPLC for thedisappearance of the starting alcohol. After the reaction was complete,it was cooled to ambient temperature, then 1.2 M NaHCO_(3(aq)) was addeduntil pH=8. The mixture was extracted with ethyl acetate (3×100 mL),then the organic phase was back extracted with brine (1×100 mL), driedover Na₂SO₄, filtered, and concentrated to dryness. The residue waspurified via silica gel chromatography, eluting with 2/1 (v/v)hexanes/ethyl acetate to give 6.0 g (55%) of the product as a lightyellow solid. MS (ESI) calcd for C₁₀H₁₂ClN₃: 209.1. found: 210.1.

Step 5. Synthesis of (9S)-tert-butyl2-chloro-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylate

(9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(1.3 g, 6.19 mmol, Note: the 5R stereochemistry is implied), Boc₂O (2.02g, 9.28 mmol, 1.5 equiv.) and DMAP (1.51 g, 12.38 mmol, 2.0 equiv.) in 5mL THF were stirred at 60° C. for 2 h. Reaction progress was monitoredby TLS and LC/MS. Water (30 mL) was added, and the mixture was extractedwith DCM (3×15 mL). The organics were concentrated and the residue waspurified via silica gel column chromatography to give (9S)-tert-butyl2-chloro-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylateas a white solid (1.3 g, 92%). MS (ESI) calcd for C₁₅H₂₀ClN₃O₂: 309.1.

Step 6. Synthesis of (9S)-tert-butyl2-(5-chloropyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylate

To a degassed mixture of dioxane/water (10 mL/1 mL) was added(9S)-tert-butyl2-chloro-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylate(650 mg, 2.096 mmol), (5-chloropyridin-3-yl)boronic acid (658 mg, 4.19mmol), Pd(dppf)Cl₂ (171 mg, 0.209 mmol), and Cs₂CO₃ (2.04 g, 6.29 mmol).The mixture was stirred at 110° C. for 12 h, then concentrated andpurified via column chromatography (PE/EA=3/1) to give (9S)-tert-butyl2-(5-chloropyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylate(600 mg, 63%). MS (ESI) calcd for C₂₀H₂₃ClN₄O₂: 386.2.

(9S)-tert-butyl2-(5-fluoropyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylatewas prepared via the same method, starting with(5-fluoropyridin-3-yl)boronic acid.

Step 7. Synthesis of(9S)-2-(5-chloropyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

This moiety was made using the following protocol. (9S)-tert-butyl2-(5-chloropyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxylate(600 mg, 1.55 mmol) was dissolved in HCl/MeOH (1M, 20 mL) and thereaction was stirred at room temp. for 1.5 h, then concentrated invacuo. Water (20 mL) and K₂CO₃ (3 g) were added, and the mixture wasstirred at room temp. for 2 h, then extracted with DCM (3×15 mL) to give(9S)-2-(5-chloropyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(450 mg, quant.). MS (ESI) calcd for C₁₅H₁₅ClN₄: 286.1.

(9S)-2-(5-fluoropyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocinewas prepared via the same method.

These moieties were used to prepare urea compounds via the general ureacoupling procedure described in the previous example.

Example 8. Preparation of(9S)-2-(5-methylpyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

This moiety was made using the following protocol. To degassed 1,4-dioxane/H₂O (20 ml, v/v=10/1) were added(9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(600 mg, 2.87 mmol), 5-methylpyridin-3-ylboronic acid (1.18 g, 3.0equiv.), PCy₃ (644 mg, 0.8 equiv.) and Pd₂(dba)₃ (330 mg, 0.2 equiv.).The mixture was heated to 110° C. in a sealed tube. After stirring 12 hrat 110° C., the black suspension was cooled to room temp andconcentrated under reduced pressure. The concentrate was suspended inEtOAc (300 ml), washed with water (4×80 ml), brine (80 ml), dried overNa₂SO₄ and concentrated under reduced pressure. The concentrate waspurified by column (DCM/MeOH=10/1) to afford the product as a lightbrown solid (756 mg, 99%). MS (ESI) calcd for C₁₆H₁₈N₄: 266.1. found:267.2 [M+H].

These conditions were also used to prepare(9S)-2-(4-methylpyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocineand4-(3-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)phenyl)morpholineby starting with the appropriate boronic acid.

The resulting moieties were used to prepare urea compounds via thegeneral urea coupling procedure described above.

Example 9. Preparation of(9S)—N-(pyridazin-3-yl)-2-(pyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

This moiety was prepared via the analogous carbamate protocol describedfor(4S)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide,using the p-chlorophenyl carbamate instead of the phenyl carbamate. MS(ESI) calcd for C₂₀H₁₉N₇O: 373.2. found: 374.3 [M+H].

Example 10. Preparation of(4S)-7-(3-chlorophenyl)-N-(6-(2,3-dihydroxypropoxy)pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To the mixture of6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrazin-2-amine (83 mg, 0.37mmol) and pyridine (29 mg, 1.37 mmol) in 3 mL of THF was addedtriphosgene (43 mg, 0.14 mmol). The above mixture was stirred at 60° C.for 2 hrs. Then(4S)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(50 mg, 0.18 mmol) was added to the reaction mixture and stirredovernight at 60° C. The crude product was purified by prep-TLC to affordthe urea intermediate as a yellow solid. To a solution of this materialin THF (3 mL) was added conc. HCl and the reaction was stirred at roomtemp. for 15 min. Sat. NaHCO₃ was added to adjust pH to 7-8. Thereaction mixture was extracted with EtOAc and the organic layer washedwith brine. Purification by prep-TLC afforded(4S)-7-(3-chlorophenyl)-N-(6-(2,3-dihydroxypropoxy)pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(8.9 mg, 40%) as a white solid. MS (ESI) calcd for C₂₃H₂₃ClN₆O₄: 482.2.found: 483.1 [M+H].

This general urea formation procedure using triphosgene could be used toprepare a variety of (4S)-7-(3-chloro or-fluorophenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby also using(4S)-7-(3-fluorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineand the appropriate amine moiety.

Example 11. Preparation of(9S)-2-(3-cyanophenyl)-N-(pyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

DIPEA (97 μL, 0.54 mmol) was added to a mixture of3-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)benzonitrile(50 mg, 0.18 mmol) and triphosgene (27 mg, 0.10 mmol) in THF (12 mL) atroom temperature. The mixture was heated at 60° C. for 30 minutes.3-aminopyridine (102 mg, 1.09 mmol) was added and the reaction mixturewas heated at reflux for 32 h. CH₃OH was added after cooling to roomtemperature. The mixture was concentrated and purified by prep HPLC. TheTFA salt was suspended in CH₃CN, 1N HCl was added and the mixturelyophilized to give(9S)-2-(3-cyanophenyl)-N-(pyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(48 mg, 61%) as the hydrochloride salt. MS (ESI) calcd for C₂₃H₂₀N₆O:396.2. found: 397.1 [M+H].

Example 12. Preparation of(9S)—N-(pyridin-2-yl)-2-(5-(trifluoromethyl)pyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

A mixture of 4-chiorophenyl pyridin-2-ylcarbamate (325 mg, 1.31 mmol),(9S)-2-(5-(trifluoromethyl)pyridin-3-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(70 mg, 0.22 mmol) and DMAP (160 mg, 1.31 mmol) in DMF (3 mL) was heatedat 100° C. in a sealed tube for 24 h. The mixture was cooled to roomtemperature then portioned between EtOAc/H₂O (60 mL/30 mL). The organiclayer was separated, washed with H₂O, brine, dried (Na₂SO₄) andconcentrated. The crude product was purified by prep-TLC (eluting withCH₂Cl₂/EtOAc/CH₃OH, 120:40:2) to afford(9S)—N-(pyridin-2-yl)-2-(5-(trifluoromethyl)pyridin-3-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamideas a tan solid (80 mg, 83%). MS (ESI) calcd for C₂₂H₁₉F₃N₆O: 440.2.found: 441.2 [M+H].

Example 13. Preparation of(4S)—N-(2-methyl-2H-indazol-5-yl)-7-(2-methylpyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-(2-methylpyridin-4-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To a solution of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]-diazepine(500 mg, 2.55 mmol) in degassed dioxane/H₂O (14 mL, v/v=10/1) was added2-methylpyridin-4-boronic acid (1.048 g, 7.65 mmol), PCy₃ (286 mg, 1.02mmol), K₃PO₄.3H₂O (1.698 g, 6.375 mmol) and Pd₂(dba)₃ (234 mg, 0.255mmol). The resulting mixture was stirred at 110° C. overnight. Themixture was cooled to room temperature then concentrated. The residuewas partitioned between EtOAc and water (50 mL each). The organic layerwas washed with water and brine, dried over Na₂SO₄, and concentrated todryness. The residue was purified by silica gel chromatography(CH₂Cl₂/THF=3/2) to give(4S)-7-(2-methylpyridin-4-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(418 mg, 82%) as a light yellow solid. MS (ESI) calcd for C₁₅H₁₆N₄:252.1. found: 253.2 [M+H].

The following intermediates were prepared using the above protocolsubstituting the appropriate boronic acid and 2-chloropyridine.2-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)benzonitrile,5-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)nicotinonitrile

Step 2. Synthesis of(4S)—N-(2-methyl-2H-indazol-5-yl)-7-(2-methylpyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of phenyl chloroformate (0.2 mL, 1.6 mmol) and pyridine(0.16 mL, 1.95 mmol) in CH₂Cl₂ (18 mL) was added2-methyl-2H-indazol-5-amine (180 mg, 1.22 mmol). The mixture was stirredat room temperature for 30 min., then quenched with sat. NaHCO₃ solution(10 mL). The aqueous phase was extracted with CH₂Cl₂ (10 mL). Thecombined organic phases were washed with brine (20 mL), dried overNa₂SO₄, and concentrated to dryness. The residue was washed with hexane(5 mL×3) to afford phenyl (2-methyl-2H-indazol-5-yl)carbamate (304 mg,93%).

A mixture of(4S)-7-(2-methylpyridin-4-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(90 mg, 0.36 mmol), phenyl (2-methyl-2H-indazol-5-yl)carbamate (285 mg,1.07 mmol) and DMAP (130 mg, 1.07 mmol) in THF (3 mL) was heated to 80°C. in a sealed tube. After heating for 36 hrs at 80° C., the mixture wasthen cooled to room tem. and concentrated. The residue was suspended inEtOAc (60 mL) and filtered. The filtrate was washed with water (20mL×3), then brine (20 mL), and dried over Na₂SO₄ and concentrated underreduced pressure. The concentrate was purified by prep-TLC(CH₂Cl₂/EtOAc/MeOH=23/1/drops) to afford(4S)—N-(2-methyl-2H-indazol-5-yl)-7-(2-methylpyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(60 mg, 40%) as a brown solid. MS (ESI) calcd for C₂₄H₂₃N₇O: 425.2.found: 426.3 [M+H].

This general urea formation procedure using phenyl carbamates could beused to prepare a variety of(4S)—N-(aryl)-7-(2-methylpyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using the appropriate amine moiety.

Example 14: Preparation of(4S)—N-(2,6-diethylphenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Under nitrogen atmosphere, to a mixture of 2,6-diethylaniline (39.1 mg,0.262 mmol, 2.0 eq) and pyridine (0.5 ml, excessive) in dry THF (3 mL)was added triphosgene (54.4 mg, 0.183 mmol). The mixture was stirred at60° C. for 2 hours and(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(40 mg, 0.131 mmol, 1.0 eq) was added to the reaction mixture andstirred for an additional 18 hours. Saturated sodium bicarbonatesolution (5 ml) and dichloromethane (10 ml) were added to the reactionmixture; the organic layer was successively washed with water (10 mL)and brine, dried (Na₂SO₄) and concentrated in vacuuo. The crude productwas purified by prep-TLC using 15:1 Ethyl Acetate in CH₂Cl₂ as eluent toafford(4S)—N-(2,6-diethylphenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidea white solid. (5.1 mg, 8% Yield). MS (ESI) calcd for C₂₇H₂₇F₃N₄O:480.21. found: 481 [M+H].

This general urea formation procedure using triphosgene could be used toprepare a variety of(4S)-7-(3-trifluoromethylphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate amine moiety for 2,6-diethylaniline.

Example 15. Preparation of(4S)-7-(2-methylpyridin-4-yl)-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-chloro-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(400 mg, 2.04 mmol), phenyl pyrazin-2-ylcarbamate (1.32 g, 6.13 mmol)and DMAP (249 mg, 2.04 mmol) in DMF (8 mL) was heated to 82° C. in asealed flask. After heating for 22 hrs, the mixture was then cooled toroom temp. and diluted with EtOAc (100 mL). The mixture was washed withwater (8 mL×9), then with brine, and dried over Na₂SO₄ and concentrated.The residue was purified by column chromatography (CH₂Cl₂/MeOH=50/1) toafford(4S)-7-chloro-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(600 mg, 93%) as a white solid. MS (ESI) calcd for C₁₄H₁₃ClN₆O: 316.1.

This general procedure could be used to prepare a variety of(4S)-7-chloro-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate carbamate moiety for phenylpyrazin-2-ylcarbamate.

Step 2. Synthesis of(4S)-7-(2-methylpyridin-4-yl)-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a mixture of(4S)-7-chloro-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(40 mg, 0.126 mmol), 2-methylpyridin-4-boronic acid (44 mg), and NaHCO₃(32 mg) in toluene/EtOH/H₂O (1.9 mL, v/v/v=10/6/3) was addedPdCl₂(PPh₃)₂ (9 mg) under a nitrogen atmosphere. The reaction mixturewas heated at reflux for 6 hrs. Another portion of2-methylpyridin-4-boronic acid (44 mg), NaHCO₃ (32 mg), and PdCl₂(PPh₃)₂(8 mg) was added and the mixture was degassed. After stirring at refluxovernight, the reaction mixture was cooled to room temp. andconcentrated. The concentrate was suspended in EtOAc (30 mL) and water(5 mL). The aqueous suspension was extracted with EtOAc (8 mL). Thecombined EtOAc phases were washed with brine (10 mL), dried over MgSO₄,and concentrated. Purification by prep-TLC (CH₂Cl₂/MeOH=50/1) afforded(4S)-7-(2-methylpyridin-4-yl)-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(18 mg, 38%) as an off-white solid. MS (ESI) calcd for C₂₀H₁₉N₇O: 373.2.found: 374.3 [M+H].

This general procedure using PdCl₂(PPh₃)₂ could be used to prepare avariety of(4S)-7-(2-methylpyridin-4-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesand(4S)-7-(6-methylpyridin-3-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using the appropriate(4S)-7-chloro-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideand the appropriate boronic acid or ester.

Example 16. Preparation of(4S)-7-(3-((R)-3-fluoropyrrolidin-1-yl)phenyl)-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-chloro-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(40 mg, 0.126 mmol),(R)-3-fluoro-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine(74 mg, 0.252 mmol), Pd(OAc)₂ (2 mg, 0.0126 mmol),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (12 mg, 0.0252mmol), and Cs₂CO₃ (82 mg, 0.252 mmol) in dioxane/H₂O (1.5 mL, v/v=9/1)was heated at 110° C. in a sealed flask. After heating overnight, themixture was cooled and filtered to remove insoluble materials. Thefiltrate was diluted with EtOAc (30 mL), washed with H₂O (5 mL×2),washed with brine (5 mL), dried over Na₂SO₄, and concentrated.Purification by prep-TLC (CH₂Cl₂/EtOAc=3/1) afforded(4S)-7-(3-((R)-3-fluoropyrrolidin-1-yl)phenyl)-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(18 mg, 32%) as a pale yellow solid. MS (ESI) calcd for C₂₄H₂₄FN₇O:445.2. found: 446.3 [M+H].

This general procedure using Pd(OAc)₂ could be used to prepare a varietyof (4S)-7-(3- and 2-substitutedphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesand(4S)-7-(2-(trifluoromethyl)pyridin-4-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using the appropriate(4S)-7-chloro-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideand the appropriate boronic acid or ester.

Example 17. Preparation of(4S)-7-(3-((S)-2,3-dihydroxypropoxy)phenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

The mixture of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(300 mg, 1.53 mmol), phenyl pyridin-2-ylcarbamate (986 mg, 4.59 mmol)and DMAP (188 mg, 1.53 mmol) in DMF (6 mL) was degassed and heated to80° C. in the sealed flask. After stirring overnight at 80° C., thereaction mixture was cooled to room temp. and partitioned into EtOAc/H₂O(150 mL/50 mL). The organic phase was washed with water (20 mL×6), brine(20 mL), dried over Na₂SO₄ and concentrated under reduced pressure. Theconcentrate was then purified by column (CH₂Cl₂/EtOAc=1/1) to afford(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(409 mg, 84%) as an off-white solid. MS (ESI) calcd for C₁₅H₁₄ClN₅O:315.1. found: 316.1 [M+H].

Step 2.(4S)-7-(3-hydroxyphenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(80 mg, 0.284 mmol), (3-hydroxyphenyl)boronic acid (78 mg, 0.568 mmol),Pd(OAc)₂ (6 mg, 0.0384 mmol),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (27 mg, 0.0568mmol) and Cs₂CO₃ (185 mg, 0.568 mmol) in dioxane/H₂O (3 mL, v/v=9/1) wasdegassed and heated at 110° C. overnight in sealed tube, then cooled toroom temp. and concentrated under reduced pressure. The concentrate wassuspended in EtOAc (20 mL), washed with water (5 mL×2), brine (5 mL),dried over Na₂SO₄, and concentrated. The residue was purified by column(CH₂Cl₂/EtOAc=1/1) to afford(4S)-7-(3-hydroxyphenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(65 mg, 61%) as an off-white solid. MS (ESI) calcd for C₂₁H₁₉N₅O₂:373.2. found: 374.2 [M+H].

Step 3. Synthesis of(4S)-7-(3-((S)-2,3-dihydroxypropoxy)phenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-hydroxyphenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(90 mg, 0.241 mmol) in DMF (3 mL) was added NaH (24 mg, 0.603 mmol).After stirring for 30 min at room temp.,(S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane (217 mg, 1.45 mmol) wasadded and the mixture was heated to 85° C. for 32 hrs in a sealed flask.The mixture was cooled to room temp. and diluted with EtOAc and washedwith water and brine. The organic phase was dried over Na₂SO₄ andconcentrated. The concentrate was dissolved in CH₂Cl₂ (3 mL) and HClsolution in dioxane (6 mL) was added. The mixture thus obtained wasstirred at room temp. for 4 hrs. The solvent was removed under reducedpressure and the concentrated was suspended in sat. NaHCO₃ (5 mL). Themixture was extracted with CH₂Cl₂ (5 mL×3) and the combined organicphases were dried over Na₂SO₄ and concentrated. Purification by prep-TLCafforded(4S)-7-(3-((S)-2,3-dihydroxypropoxy)phenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(24 mg, 33%) as an off-white powder. MS (ESI) calcd for C₂₄H₂₅N₅O₄:447.2. found: 448.3 [M+H].

This general procedure could be used to prepare a variety of(4S)-7-(3-((S)-2,3-dihydroxypropoxy)phenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using the appropriate(4S)-7-(3-hydroxyphenyl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.

Example 18. Preparation of(4S)-7-(1-propyl-1H-pyrazol-4-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-chloro-N-(pyradyl-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(999 mg, 3.26 mmol, 1.0 eq) as combined with DIEA (1.7 mL, 9.78 mmol,3.0 eq) into methylene chloride (30 mL) and cooled to 0° C. on anicebath. Triphosgene (482 mg, 1.63 mmol, 0.5 eq) was then added inseveral small portions to the stirring solution. The icebath was theremoved and the reaction was allowed to warm to room temperature. Thereaction was then left stirring overnight. Pyridine-3-amine (800 mg,3.60 mmol, 1.1 eq) was then added slowly in small portions over severalminutes. The mixture was then stirred at room temperature for 2 hours.The mixture was then treated with water (100 mL) diluted with EtOAc (100mL). Phases were separated and the organic phase was dried over Na₂SO₄and concentrated. The residue was purified by column chromatographyusing a gradient of 15-100% (EtOAc/Pentane) to afford(4S)-7-chloro-N-(pyradyl-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(761 mg, yield 47%). MS (ESI) calcd for C₁₅H₁₄ClN₅O 315.1. found: 315.7[M+H].

Step 2.(4S)-7-(1-propyl-1H-pyrazol-4-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

The title compound was prepared using the following protocol: A mixtureof(4S)-7-chloro-N-(pyradyl-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(100 mg, 0.32 mmol, 1.0 eq), 1-ethyl-1H-pyrazole-4-boronic acid (151 mg,0.64 mmol, 2.0 eq), Pd(dppf)Cl₂ (26.7 mg, 0.06 mmol, 0.2 eq) and Cs₂CO₃(208 mg, 0.64 mmol, 2.0 eq) in dioxane/H₂O (5 mL) was stirred at 100° C.for 6 h. Water was added and the mixture was extracted with EtOAc. Theorganics were dried over anhydrous Na₂SO₄, concentrated and purified bypre-TLC to give(4S)-7-(1-propyl-1H-pyrazol-4-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.(37.1 mg, yield 30%) MS (ESI) calcd for C₂₁H₂₃N₇O 389.2. found: 390.3[M+H].

This general procedure using Pd(dppf)Cl₂ could be used to prepare avariety of(4S)-7-(pyridin-3-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate boronic acid or ester moiety for2,2-difluorobenzo[d][1,3]dioxole-5-boronic acid.

Example 19: Preparation of(4S)-7-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of3-(Pyridin-2-yl)-2H-pyrido[1,2-a][1,3,5]triazine-2,4(3H)-dione

The title compound was prepared using the following protocol: 10.0 g ofpicolinic acid (81.2 mmol, 1 eq) was suspended in 250 mL of Toluene.20.0 mL of diphenyl phosphoryl azide (92.6 mmol, 1.14 eq) was added.13.4 mL of triethylamine (95.8 mmol, 1.18 eq) was added dropwise. Thereaction mixture was stirred at room temperature for 30 minutes, thenfor 2 hrs at 80° C. The reaction mixture was allowed to cool to roomtemperature. Solids were filtered and washed with ethyl acetate andpentane. The solid was dried under high vacuum. Obtained 6.46 g (66%yield) of a brown solid. MS (ESI) calcd for C₁₅H₁₄ClN₅O: 240.06. found:241.31 [M+H].

This general procedure could be used to prepare3-(Pyridin-2-yl)-2H-pyrido[1,2-a][1,3,5]triazine-2,4(3H)-dione and3-(pyrazin-2-yl)-2H-pyrazino[1,2-a][1,3,5]triazine-2,4(3H)-dione bysubstituting the appropriate heteroaryl carboxylic acid bearing nitrogenheteroatom in the 2 position on the six membered aromatic ring.

Step 2. Synthesis of(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

The title compound was prepared using the following protocol:(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(2.0 g, 10.2 mmol, 1.0 eq) was dissolved in 2-methyl-tetrahydrofuran (40mL) and treated with NaH (1.7 g, 30.6 mmol, 3.0 eq) at room temperature.The resulting mixture was then stirred at RT for 30 minutes.3-(pyridin-2-yl)-2H-pyrido[1,2-a][1,3,5]triazine-2,4(3H)-dione (2.4 g,10.2 mmol, 1.0 eq) was then added and the reaction was then fitted witha reflux condenser and heated to 80° C. overnight. The reaction was thencooled RT, placed on an icebath, and quenched with slow addition ofNaHCO₃ (65 mL). Crude reaction was then extracted 3×'s EtOAc (75 mLeach) and organics were dried over anhydrous MgSO₄ and concentrated.Reaction was purified via column chromatography using a gradient of10-100% EtOAc/Pentane to give(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(2.5 g, 78%). MS (ESI) calcd for C₁₅H₁₄ClN₅O: 315.09. found: 316.10[M+H].

Step 3. Synthesis of(4S)-7-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(79 mg, 0.250 mmols, 1.0 eq) was combined with Pd₂(dba)₃ (2.3 mg, 0.006mmols, 0.02 eq), K₃PO₄ (80 mg, 0.380 mmols, 2 eq), S-Phos (4.8 mg, 0.012mmol, 0.05 eq) and the flask was purged with N₂ and sealed. N-butanol (1mL) was then added via syringe and the reaction was heated to 100° C.for 3 hrs. Reaction was then cooled to room temperature filtered thenpurified directly via reverse phase chromatography using a gradient of5-95% CH₃CN/H₂O (0.1% TFA) to give(4S)-7-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(11 mg, 10%). C₁₅H₁₄ClN₅O: 437.13. found: 438.17 [M+H].

This general procedure using Pd(dppf)Cl₂ could be used to prepare avariety of(4S)-7-(pyridin-2-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesand(4S)—N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideby substituting the appropriate boronic acid or ester moiety for2,2-difluorobenzo[d][1,3]dioxole-5-boronic acid.

Example 20. Preparation of(4S)—N-(5-fluoropyridin-2-yl)-7-(2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-chloro-N-(5-fluoropyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(280 mg, 1.43 mmol), 4-chlorophenyl (5-fluoropyridin-2-yl)carbamate(1.14 g, 4.29 mmol) and DMAP (174 mg, 1.43 mmol) in DMF (7 mL) washeated to 80° C. in a sealed flask. After stirring overnight at 80° C.,the reaction mixture was cooled to room temp. and diluted with EtOAc(100 mL). The organic phase was washed with water (50 mL×1, 10 mL×6),then brine (50 mL), and dried over Na₂SO₄ and concentrated. Theconcentrate was then purified by column (CH₂Cl₂/EtOAc=1/1) to afford(4S)-7-chloro-N-(5-fluoropyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(360 mg, 75%) as an off-white solid. MS (ESI) calcd for C₁₅H₁₃ClFN₅O:333.1.

Step 2. Synthesis of(4S)—N-(5-fluoropyridin-2-yl)-7-(2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-chloro-N-(5-fluoropyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(50 mg, 0.149 mmol),4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyridine(102 mg, 0.298 mmol), PCy₃ (8 mg, 0.0298 mmol), Pd₂(dba)₃ (14 mg, 0.0149mmol), and K₃PO₄.3H₂O (79 mg, 0.373 mmol) in degassed dioxane/H₂O (2 mL,v/v=9/1) was heated to 120° C. in a sealed flask. After stirringovernight, the mixture was cooled to room temperature and diluted withEtOAc (60 mL). The diluted solution was washed with H₂O (20 mL×1, 10mL×5) and brine (20 mL), dried over Na₂SO₄ and concentrated.Purification by prep-TLC (CH₂Cl₂/EtOAc/MeOH=3/1/2 drops) afforded(4S)—N-(5-fluoropyridin-2-yl)-7-(2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyridin-4-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(54 mg, 71%) as an off-white solid. MS (ESI) calcd for C₂₅H₂₃F₄N₇O:513.2. found: 514.3 [M+H].

This general procedure using Pd₂(dba)₃ could be used to prepare avariety of (4S)-7-(2-(3-substituted-pyrrolidin-1-yl)pyridin-4-yl)-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby using the appropriate(4S)-7-chloro-N-(aryl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideand the appropriate boronic acid or ester.

Example 21. Preparation of(4S)-7-(3-chlorophenyl)-9-methoxy-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of 2,6-dichloro-4-methoxypyridine

To a solution of 2,4,6-trichloropyridine (30 g, 165 mmol) in MeOH wasslowly added sodium methoxide (10.7 g, 197 mmol). The mixture wasstirred overnight and quenched with 300 ml of water. The suspension wasfiltered, washed with water and petroleum ether to obtain2,6-dichloro-4-methoxypyridine as a white solid (18.0 g, 61% yield). MS(ESI) calcd for C₆H₅Cl₂NO: 176.97.

Step 2. Synthesis of 2,6-dichloro-4-methoxy-3-nitropyridine

To a solution of 2,6-dichloro-4-methoxypyridine (18.1 g, 102 mmol) insulfuric acid (110 mL) was added nitric acid (15.6 mL) dropwise at 0°C., and then the mixture was heated to 100° C. for 2 hours. The reactionmixture was poured into ice-water, the suspension was filtered andwashed with water to obtain 2,6-dichloro-4-methoxy-3-nitropyridine as awhite solid (19.9 g, 88% yield). MS (ESI) calcd for C₆H₄Cl₂N₂O₃: 221.96.

Step 3. Synthesis of (S)-di-tert-butyl2-((6-chloro-4-methoxy-3-nitropyridin-2-yl)amino)succinate

To a solution of 2,6-dichloro-4-methoxy-3-nitropyridine (14.5 g, 65mmol) and (S)-1,4-di-tert-butoxy-1,4-dioxobutan-2-aminium chloride (22g, 78 mmol) in DMF (150 mL) was added DIEA (32.3 mL), and the mixturewas heated to 80° C. for 3 hours. DMF was removed under vacuum and theresidue was dissolved in ethyl acetate, washed with brine, dried overanhydrous Na₂SO₄ and purified by flash chromatography (10% Ethyl Acetatein Petroleum Ether) to obtain (S)-di-tert-butyl2-((6-chloro-4-methoxy-3-nitropyridin-2-yl)amino)succinate as a yellowoil (4.8 g, 16% yield). MS (ESI) calcd for C₁₈H₂₆ClN₃O₇: 431.15.

Step 4. Synthesis of (S)-tert-butyl2-(6-chloro-8-methoxy-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate

To a mixture of (S)-di-tert-butyl2-((6-chloro-4-methoxy-3-nitropyridin-2-yl)amino)succinate (4.7 g, 10.9mmol) in AcOH (60 ml) was added iron powder (6.107 g, 109 mmol) and thereaction mixture was stirred at 100° C. for 2 hours. The reaction wasquenched with 1 N NaOH, and extracted with ethyl acetate. The organicswere washed with brine and purified by flash chromatography (40% ethylacetate in petroleum ether) to obtain (S)-tert-butyl2-(6-chloro-8-methoxy-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetateas a yellow oil (1.87 g, 52% yield). MS (ESI) calcd for C₁₄H₁₈ClN₃O₄:327.10.

Step 5. Synthesis of(S)-2-(6-chloro-8-methoxy-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol

To a solution of (S)-tert-butyl2-(6-chloro-8-methoxy-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)acetate(1.7 g, 5.2 mmol) in THF (20 mL) was added BH₃-Me₂S (5.2 mL, 10 M inMe₂S, 52 mL), and the reaction mixture was then heated at 50° C.overnight. Upon cooling to room temperature, the reaction was quenchedwith dropwise addition of water, then 1N aqueous HCl (10 mL) was addedand the mixture was stirred at 50° C. for 2 hours. Saturated NaHCO₃ wasadded and the mixture was extracted with CH₂Cl₂, and concentrated to anoil. The oil was treated with TFA (15 mL) in CH₂Cl₂ (15 mL) for 2 hoursand the DCM and TFA were removed under vacuum. The residue was dissolvedin MeOH (20 mL) and Cs₂CO₃ (2 g) was added. The mixture was stirred for1 hour, concentrated and purified by flash chromatography (30:1CH₂Cl₂/MeOH) to obtain(S)-2-(6-chloro-8-methoxy-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanolas a yellow oil (997 mg, 79% yield). MS (ESI) calcd for C₁₀H₁₄ClN₃O₂:243.08.

Step 6. Synthesis of(4S)-7-chloro-9-methoxy-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To a solution of PPh₃ (1.003 g, 3.83 mmol) in CH₂Cl₂ (50 mL) was addedDDQ (869 mg, 3.83 mmol), then(S)-2-(6-chloro-8-methoxy-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethanol(620 mg, 2.55 mmol) was added. The mixture was stirred for 30 min,concentrated and purified by flash chromatography (33 to 100% Ethylacetate in Petroleum Ether) to obtain(4S)-7-chloro-9-methoxy-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineas a yellow solid (413 mg, 72% yield). MS (ESI) calcd for C₁₀H₁₂ClN₃O:225.07.

Step 7. Synthesis of(4S)-7-(3-chlorophenyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of(4S)-7-chloro-9-methoxy-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(226 mg, 1.0 mmol, (3-chlorophenyl)boronic acid (187 mg, 1.2 mmol),Cs₂CO₃ (654 mg, 2.0 mmol) and Pd(dppf)Cl₂.DCM (40 mg, 0.05 mmol) in 10:1Dioxane/Water (6 mL) solution was microwave heated (130° C.×1 h). Thereaction mixture was concentrated to dryness, suspended in CH₂Cl₂,washed with sat. NaHCO₃, water, brine, dried over MgSO₄ andconcentrated. The reaction mixture was initially purified by silica gelchromatography (0 to 10% MeOH in CH₂Cl₂ gradient) and subsequentlypurified by Prep HPLC. The reaction was repeated a 2^(nd) time at thesame scale, and the combined HPLC fractions were lyophilized to obtain(4S)-9-methoxy-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(199 mg, 33% yield). MS (ESI) calcd for C₁₆H₁₆ClN₃O: 301.10. found: 302[M+H].

This general procedure could be used to prepare(4S)-9-methoxy-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineby substituting (3-(trifluoromethyl)phenyl)boronic acid for(3-chlorophenyl)boronic acid.

Step 8. Synthesis of(4S)-7-(3-chlorophenyl)-9-methoxy-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-chlorophenyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(60 mg, 0.2 mmol) in THF (10 mL) was added 60% NaH suspension in mineraloil (24 mg, 1 mmol). The mixture was heated to reflux for 1 hour,3-(pyrazin-2-yl)-2H-pyrazino[1,2-a][1,3,5]triazine-2,4(3H)-dione (73 mg,0.3 mmol) was added and the mixture was heated at reflux for anadditional 2 hours. The reaction mixture was cooled to room temperature,concentrated to dryness, diluted with sat. NaHCO₃, and extracted withCH₂Cl₂ (3×). The organics were washed with Brine, dried (Na₂SO₄),concentrated and purified by prep-HPLC and lyophilized to afford(4S)-7-(3-chlorophenyl)-9-methoxy-N-(pyrazin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(54 mg, 64% yield). MS (ESI) calcd for C₂₁H₁₉ClN₆O₂: 422.13. found: 423[M+H].

Example 22. Preparation of(4S)-9-methoxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of the phenyl pyridin-2-ylcarbamate (191.6 mg, 0.8995 mmol),(4S)-9-methoxy-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(150 mg, 0.4477 mmol) and DMAP (65.55 mg, 0.5373 mmol) in 15 ml ofacetonitrile were stirred at 60° C. overnight. The mixture was directlyloaded on prep-TLC and purified (using ethyl acetate as eluent) toafford(4S)-9-methoxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a white solid (150 mg, Yield: 74%). MS (ESI) calcd for C₂₃H₂₀F₃N₅O₂:455.2. found: 456 [M+H].

This general procedure could be used to prepare(4S)-9-methoxy-N-(Aryl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate carbamate moiety for phenylpyridin-2-ylcarbamate.

Example 23. Preparation of(4S)-9-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Under nitrogen atmosphere, to the mixture of(4S)-9-methoxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(50 mg, 0.1098 mmol) in 3 ml of dry CH₂Cl₂ was added BBr₃ (0.5 mL,0.5494 mmol) dropwise at 0° C. Then reaction mixture was stirred at 50°C. overnight. Sodium bicarbonate solution (5 mL) and dichloromethane (10mL) were added to the reaction mixture and the organic layer was washedwith water, brine, dried with anhydrous sodium sulfate and concentratedin vacuuo. The crude product was purified by prep-TLC using (1:20 MeOHin CH₂Cl₂) as eluent to afford(4S)-9-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a white solid (18 mg, 35% yield). MS (ESI) calcd for C₂₂H₁₈F₃N₅O₂:441.1. found: 442 [M+H].

This general procedure could be used to prepare((4S)-9-hydroxy-N-(Aryl)-7-(3(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate(4S)-9-methoxy-N-(Aryl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidefor(4S)-9-methoxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.

Example 24. Preparation of(4S)—N-(4-((3-(3-methyl-3H-diazirin-3-yl)propanamido)methyl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of(4S)—N-(4-(aminomethyl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidehydrochloride (49 mg, 0.1 mmol), 2,5-dioxopyrrolidin-1-yl3-(3-methyl-3H-diazirin-3-yl)propanoate (23 mg, 0.1 mmol), andtriethylamine (70 μL, 0.5 mmol) in DMF (2 mL) was stirred at roomtemperature for 1 hour. Water (10 mL) and sat. NaHCO₃ (5 mL) was added,and the reaction mixture was extracted with CH₂Cl₂ (3×) and concentratedto dryness. The crude product was purified on silica gel chromatography(0 to 10% MeOH gradient in CH₂Cl₂), concentrated, chased withdiethylether and pentane, and dried under vacuum to afford(4S)—N-(4-((3-(3-methyl-3H-diazirin-3-yl)propanamido)methyl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a white foam (39 mg, 68% yield). MS (ESI) calcd for C₂₉H₂₈F₃N₇O₂:563.23. found: 564 [M+H].

Example 25: Preparation of(4S)—N-(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(46 mg, 0.15 mmol) and triphosgene (36 mg, 120 mmol) in CH₂Cl₂ (2 mL)was added triethylamine (56 μL, 0.45 mmol). The reaction mixture wasstirred at 40° C. for 2.5 hours and3-(3-(trifluoromethyl)-3H-diazirin-3-yl)aniline (40 mg, 0.2 mmol)dissolved in CH₂Cl₂ (1 mL) was added. The reaction mixture was stirredat room temperature for 2 hours. The organic layer was washed with Sat.NaHCO₃, water, brine dried over Na₂SO₄ and conc. The residue waspurified by flash chromatography (eluting first with a 0 to 100% CH₂Cl₂gradient in pentane, then 0 to 10% MeOH in CH₂Cl₂) to afford(4S)—N-(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(40 mg, 50% yield). MS (ESI) calcd for C₂₅H₁₈F₆N₆O: 532.14. found: 533[M+H].

The above 3-(3-(Trifluoromethyl)-3H-diazirin-3-yl)aniline was preparedaccording to Biasotti B. et. al., Bioorganic and Medicinal Chemistry,2003, 11, 2247-2254.

This general procedure was used to prepare a variety of(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)ureas by substituting(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepinewith the appropriate amine.

Example 26. Preparation of(3R,4R)-7-chloro-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineStep 1. Synthesis of (S)-dimethyl 2-benzamidosuccinate

To a 5 L three-necked flask, equipped with a thermometer, a condenser,and a mechanical stirrer was added 161 g (1.00 mol) of L-aspartic aciddimethyl ester, 2500 mL of dichloromethane, and 198 g (1.96 mol) oftriethylamine. The solution was cooled to −5° C., then 156 g (1.11 mol)of benzoyl chloride was added, dropwise, keeping the internaltemperature at −5° C. The mixture was stirred at −5° C. for 1 h, then itwas filtered. The precipitate was washed three times with additionaldichloromethane, then the combined filtrate and washings were extractedwith saturated aqueous K₂CO₃ solution. The dichloromethane layer wasdried over Na₂SO₄ and then concentrated in vacuo to give 200 g (75%) ofthe product as a white solid. MS (ESI) calcd for C₁₃H₁₅NO₅: 265.1.

Step 2. Synthesis of (4S,5S)-dimethyl2-phenyl-4,5-dihydrooxazole-4,5-dicarboxylate

To a 10 L four necked flask, equipped with a thermometer, a mechanicalstirrer, and a N₂ inlet was added 100 g (0.377 mol) of (S)-dimethyl2-benzamidosuccinate, then 4 L of dry tetrahydrofuran. The mixture wasstirred and cooled to 0° C. To the solution was added 770 mL (0.77 mol)of a 1.0 M solution of lithium bis(trimethylsilyl)amide intetrahydrofuran, keeping the internal temperature at 0° C. during theaddition. The reaction was stirred at 0° C. for 30 min, then it wascooled to −78° C. To this was added a solution of 195 g (0.77 mol) ofiodine in 2 L of tetrahydrofuran, dropwise, at −78° C. The reaction wasstirred at −78° C. for 1 h, then it was quenched by the addition of 2 Lof saturated NH₄Cl_((aq)), and 400 g (2.53 mol) of Na₂S₂O₃. The mixturewas stirred at ambient temperature for 30 min, then 2 L of ethyl acetatewas added, and the layers were separated. The aqueous phase wasextracted with additional ethyl acetate (3×2 L). The combined ethylacetate layers were dried over Na₂SO₄ and then concentrated in vacuo.The residue was purified via silica gel chromatography, eluting with20:1 (v/v) heptanes: ethyl acetate to give 30 g (30%) of the product asa white solid. MS (ESI) calcd for C₁₃H₁₃NO₅: 263.1.

Step 3. Synthesis of (2S,3S)-2-amino-3-hydroxysuccinic acid

To a 500 mL flask equipped with a reflux condenser was added 13 g (50mmol) of (4S,5S)-dimethyl 2-phenyl-4,5-dihydrooxazole-4,5-dicarboxylate,and 200 mL (2.4 mol) of 12M HCl_((aq)). The reaction was stirred at 50°C. for 16 h, then the solvent was removed in vacuo. The residue wastaken up in 1000 mL of water, and extracted with ethyl acetate until nobenzoic acid was present in the aqueous layer by HPLC. The organiclayers were discarded, and the aqueous layer was concentrated in vacuoto give 8.6 g (94%) of (2S,3S)-2-amino-3-hydroxysuccinic acidhydrochloride as a white crystalline solid. MS (ESI) calcd for C₄H₇NO₅:149.0.

Step 4. Synthesis of (2S,3S)-dimethyl 2-amino-3-hydroxysuccinate.0

To a 500 mL three-necked flask equipped with a reflux condenser wasadded 170 mL of methanol. The methanol was cooled to −5° C., then 23.6 g(198 mmol) of SOCl₂, was added dropwise. After the addition wascomplete, 8.6 g (46 mmol) of (2S,3S)-2-amino-3-hydroxysuccinic acid HClsalt was added, and the solution was stirred at ambient temperature for16 h. The solvent was removed in vacuo, to give crude (2S,3S)-dimethyl2-amino-3-hydroxysuccinate HCl salt as a yellow oil, which was usedwithout further purification in the next step. MS (ESI) calcd forC₆H₁₁NO₅: 177.1.

Step 5. Synthesis of (2S,3S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate

To a 500 mL round-bottomed flask equipped with a reflux condenser wasadded 18 g (84.3 mmol) of (2S,3S)-dimethyl 2-amino-3-hydroxysuccinateHCl, 29 g (150 mmol) of 2,6-dichloro-3-nitropyridine, 42.5 g NaHCO₃ (506mmol), and 350 mL of THF. The reaction was stirred at 40° C. for 36 h.The solids were filtered away and washed with additional THF (30 mL×3).The filtrate and washings were combined and concentrated in vacuo. Theresidue was purified via silica gel chromatography, eluting with agradient of 5:1 (v/v) to 1:1 (v/v) heptanes: ethyl acetate to give 22 g(63%) of (2S,3S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate as a yellowcrystalline solid. MS (ESI) calcd for C₁₁H₁₂ClN₃O₇: 333.0.

This procedure could be used to prepare dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate bysubstituting dimethyl 2-amino-3-hydroxysuccinate hydrochloride for(2S,3S)-dimethyl 2-amino-3-hydroxysuccinate hydrochloride.

Step 6. Synthesis of (S)-methyl2-((S)-6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)-2-hydroxyacetate

A slurry of 10 g Raney Ni in H₂O was decanted to remove the water,diluted with 2-propanol and decanted again to give a wet mixtureweighing 10 g. To a 500 mL flask was added 10 g (30 mmol) of(2S,3S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate, 200 mL of2-propanol, then 10 g of Raney Ni. The reaction was put under vacuum andback filled with hydrogen 3 times, then it was stirred under 1 atm. ofH₂ for 3 h, or until no starting nitro compound remained by HPLC. TheRaney Ni was filtered away, then the filtrate was put into a 500 mLround bottomed flask, and 5 mL (87 mmol) of glacial acetic acid wasadded. The flask was fitted with a reflux condenser, then the reactionwas stirred at 80° C. for 16 h, until no intermediate diaminopyridinewas present by HPLC. The solvents were removed in vacuo. The residue waspurified via silica gel chromatography, eluting with 5/1 (v/v)heptanes/ethyl acetate to give 6 g (72%) of the product as a lightyellow solid. MS (ESI) calcd for C₁₀H₁₀ClN₃O₄: 271.0.

This procedure could be used to prepare methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)-2-hydroxyacetateby substituting dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate for(2S,3S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)-3-hydroxysuccinate.

Step 7. Synthesis of(S)-1-((R)-6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethane-1,2-diol

To a 100 mL 3-necked round bottomed flask equipped with a refluxcondenser and a thermometer was added 20 mL of tetrahydrofuran, then1.19 g (30 mmol) of LiAlH₄. The stirred mixture was cooled to −5° C.,then a solution of 0.5 g (2 mmol) of (S)-methyl2-((S)-6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)-2-hydroxyacetatein 20 mL of tetrahydrofuran was added dropwise. The reaction was stirredat 70° C. for 16 h, or until the reaction was complete by HPLC. (Lactamreduction was slower than ester reduction.) The reaction was cooled to−10° C., then 1.2 mL of water was added, dropwise, and the reaction wasstirred for 10 min. Next, 1.2 mL of 15% (w/v) NaOH(aq.) was addeddropwise, and the reaction was stirred for 20 min. To complete thequench of the excess LiAlH₄, another 3.6 mL of water was added dropwise,then the reaction was stirred for 20 min. The reaction was filtered, andthe precipitate was washed with tetrahydrofuran (3×20 mL). The combinedfiltrate and washings were concentrated in vacuo to give about 1.5 g ofa solid. This was diluted with 16 mL of ethyl acetate and filtered. Thefiltrate was concentrated in vacuo to give 310 mg (80%) of the productas a brown solid. MS (ESI) calcd for C₉H₁₂ClN₃O₂: 229.1.

This procedure could be used to prepare1-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethane-1,2-diolby substituting methyl2-((S)-6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)-2-hydroxyacetatefor (S)-methyl2-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)-2-hydroxyacetate.

Step 8. Synthesis of(1S,4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol

To a 10 mL round bottomed flask equipped with a reflux condenser wasadded 250 mg (1.1 mmol) of(S)-1-((R)-6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethane-1,2-diol,then 5 mL of 48% HBr_((aq)). The reaction was heated at 105° C. for 16h, or until HPLC showed that all of the starting material had beenconsumed. The reaction was cooled, then K₂CO_(3(S)) was slowly addeduntil pH=8. The solvent was removed in vacuo, then the residue waspurified via silica gel chromatography, eluting with 20/1 (v/v)dichloromethane/methanol to give 110 mg (47%) of the product as a whitecrystalline solid. MS (ESI) calcd for C₉H₁₀ClN₃O: 211.1.

This procedure could be used to prepare7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-olby substituting6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethane-1,2-diol for(S)-1-((R)-1-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)ethane-1,2-diol.

Step 9. Synthesis of(1S,4R)-7-chloro-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To a 10 mL round bottomed flask was added 1.68 g (7.9 mmol) of(1S,4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol,5 mL of N,N-dimethylformamide, and 2.8 mL (24 mmol) of2,6-dimethylpyridine. The mixture was stirred until it was homogeneous,then 1.5 mL (12 mmol) of chlorotrimethylsilane was added, dropwise, atambient temperature. The reaction was stirred at ambient temperature for3 h, then it was diluted with 100 mL of dichloromethane, and extractedwith saturated NaHCO_(3(aq)) (1×50 mL), then brine (3×50 mL), andconcentrated in vacuo to give 2.04 g (91%) of the product as a whitecrystalline solid. MS (ESI) calcd for C₁₂H₁₈ClN₃OSi: 283.1.

This procedure could be used to prepare7-chloro-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineby substituting7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-olfor(1S,4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol.

Example 27. Preparation of(3R,4R)-7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of(3R,4R)-7-chloro-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(283 mg, 1.0 mmol), (3-(trifluoromethyl)phenyl)boronic acid (285 mg, 1.5mmol), XPhos (24 mg, 0.05 mmol), Pd(OAc)₂ (5.6 mg, 0.025 mmol), Cs₂CO₃(977 mg, 3.0 mmol) in 10:1 dioxane:water (8.8 mL) was degassed andmicrowave heated at 100° C. for 25 min. The dioxane layer wasconcentrated and purified by flash chromatography (0 to 7% MeOH gradientin CH₂Cl₂) to obtain(3R,4R)-7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine.The fractions were concentrated, dissolved in EtOAc, washed with sat.NaHCO₃, water, brine, dried (Na₂SO₄) and concentrated to obtain(3R,4R)-7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(338 mg, 85% yield). MS (ESI) calcd for C₁₉H₂₂F₃N₃OSi: 393.15. found:394 [M+H].

This general coupling procedure using Sodium hydride could be used toprepare(3R,4R)-3-hydroxy-N-aryl-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideby substituting the appropriate aryl isocyanate or aryl isocyante dimerfor 3-(pyridin-2-yl)-2H-pyrido[1,2-a][1,3,5]triazine-2,4(3H)-dione. Thenon-stereospecific series could be made starting with the7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine.

Example 28. Preparation of(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of(3R,4R)-7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(215 mg, 0.55 mmol) and 60% NaH in mineral oil (66 mg, 1.65 mmol) in THF(30 mL) was heated to reflux for 20 min.3-(pyridin-2-yl)-2H-pyrido[1,2-a][1,3,5]triazine-2,4(3H)-dione (197 mg,0.82 mmol) was added and the reaction mixture was heated at reflux for 2hours. The reaction mixture was cooled, concentrated to dryness, dilutedwith CH₂Cl₂. The organic layer was washed with sat. NaHCO₃, water, brinedried (Na₂SO₄) and concentrated to dryness. The residue was purified byPrep-HPLC, and the fractions were concentrated to dryness and trituratedwith a mixture of diethyl ether and pentane to obtain(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide2,2,2-trifluoroacetate as a white solid (132 mg, 44% yield). MS (ESI)calcd for C₂₂H₁₈F₃N₅O₂: 441.14. found: 442 [M+H].

Example 29. Preparation of(4R)-3-oxo-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide2,2,2-trifluoroacetate (92 mg, 0.166 mmol) in CH₂Cl₂ (20 mL) was addedDess-Martin Periodane (105 mg, 0.25 mmol). The reaction mixture wasstirred at room temperature for 1.5 hours. A second aliquot ofDess-Martin-Periodane (105 mg, 0.25 mmol) was charged and the reactionmixture was stirred at room temperature for 0.5 hours. A solution ofSat. NaHCO₃ (aq) was added and the reaction mixture was extracted withCH₂Cl₂. The organic layer was washed with brine, dried (Na₂SO₄), andconcentrated to a white foam. The residue was purified by flashchromatography (0 to 100% Ethyl Acetate in Penate), and then purified byPrep-HPLC and lyophilized to obtain(4R)-3-oxo-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide2,2,2-trifluoroacetate (62 mg, 67% yield). MS (ESI) calcd forC₂₂H₁₆F₃N₅O₂: 439.13. found: 440 [M+H].

This general procedure was used to prepare(4R)—N-(3-(oxazol-5-yl)phenyl)-3-oxo-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideby substituting(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidewith(3R,4R)-3-hydroxy-N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Example 30. Preparation of(3S,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4R)-3-oxo-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide2,2,2-trifluoroacetate (50 mg, 0.09 mmol) in THF (10 mL) at −78° C.,under nitrogen atmosphere, was dropwise added a solution of 1 MSuperHydride in THF (0.45 mL, 0.45 mmol). The reaction mixture wasstirred at −78° C. for 30 min, quenched with the addition of EtOAc (5mL), warmed to room temperature and concentrated. The residue waspurified by flash chromatography (0 to 10% MeOH gradient in CH₂Cl₂) toafford(3S,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(22 mg, 55% yield). MS (ESI) calcd for C₂₂H₁₈F₃N₅O₂: 441.14. found: 442[M+H].

Example 31. Preparation of(3S,4R)-5-(pyridin-2-ylcarbamoyl)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ylacetate

To a solution of(3S,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(15 mg, 0.034 mmol) in CH₂Cl₂ was added triethylamine (10 μL, 0.07 mmol)followed by DMAP (1 mg) and acetic anhydride (10 μL, 0.011 mmol). Thereaction mixture was stirred at room temperature for 2 hours,concentrated to dryness and purified by Prep-HPLC. The fractions werelyophilized to obtain(3S,4R)-5-(pyridin-2-ylcarbamoyl)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ylacetate 2,2,2-trifluoroacetate (5.9 mg, 29% yield). MS (ESI) calcd forC₂₄H₂₀F₃N₅O₃: 483.15. found: 484 [M+H].

This general procedure was used to prepare(3R,4R)-5-(pyridin-2-ylcarbamoyl)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ylacetate by substituting(3S,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidewith(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.

This general procedure was used to prepare(3R,4R)-5-(pyridin-2-ylcarbamoyl)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ylbenzoate substituting(3S,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidewith(3R,4R)-3-hydroxy-N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide,and substituting acetic anhydride and benzoic anhydride.

Example 32. Preparation of(3R,4R)-3-hydroxy-N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of(3R,4R)-7-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(284 mg, 0.722 mmol), phenyl (3-(oxazol-5-yl)phenyl)carbamate (404 mg,1.44 mmol) and DMAP (44 mg, 0.36 mmol) in CH₃CN (20 mL) was heated at60° C. (overnight) and then 80° C. (2 h). Then to the reaction mixturewas added an additional quantity of phenyl(3-(oxazol-5-yl)phenyl)carbamate (202 mg, 0.72 mmol) and DMAP (88 mg,0.72 mmol). The reaction mixture was heated at 80° C. overnight andconcentrated to dryness. The reaction mixture was initially purified bycolumn chromatography (0 to 100% ethyl acetate in pentane gradient) andthen purified by prep-HPLC and lyophilized to obtain(3R,4R)-3-hydroxy-N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas the trifluoroacetic acid salt (161 mg, 36%). MS (ESI) calcd forC₂₆H₂₀F₃N₅O₃: 507.15. found: 508 [M+H].

Example 33. Preparation of(3R,4R)-7-(3-chlorophenyl)-3-hydroxy-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

TBSOTf (111 mg, 0.42 mmol) was added slowly to a solution of(3R,4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol(90 mg, 0.28 mmol) in 2 mL of CH₂Cl₂ under N₂ at −20° C. The mixture wasstirred for 1 h then washed with 1N HCl and water, dried over anhydrousNa₂SO₄ and evaporated to dryness. The residue was purified by Prep.TLC(DCM/EA=20:1) to give(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineas a yellow solid (90 mg, 73% yield), MS (ESI) calcd for C₁₅H₂₄ClN₃OSi:325.14;

Step 2. Synthesis of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(1.08 g, 3.32 mmol), (3-chlorophenyl)boronic acid (570 mg, 3.65 mmol),CS₂CO₃ (2.48 g, 7.63 mmol), Pd(dppf)Cl₂ (300 mg, 0.33 mmol) indioxane/H₂O (11 mL, 10:1) was heated at 130° C. for 2.5 h in a microwavereactor. The mixture was poured into water, and diluted with EtOAc. Theorganic phase washed with water and brine, dried over anhydrous Na₂SO₄,and evaporated to dryness. The residue was purified by prep. HPLC togive(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineas a light yellow solid (850 mg, 63% yield), MS (ESI) calcd forC₂₁H₂₈ClN₃OSi: 401.17;

This general procedure was used to prepare(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(5-(trifluoromethyl)pyridin-3-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine.

Step 3. Synthesis of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of phenyl pyridine-2-ylcarbamate (86 mg, 0.20 mmol),(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(80 mg, 0.20 mmol) and DMAP (24 mg, 0.20 mmol) in 5 ml of MeCN wasstirred at 65° C. overnight. The crude reaction mixture was purified byprep. TLC eluting with DCM:EA=20:1 to give(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(110 mg). MS (ESI) calcd for C₂₇H₃₂ClN₃O₂Si: 521.2;

Step 4. Synthesis of(3R,4R)-7-(3-chlorophenyl)-3-hydroxy-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(110 mg, 0.21 mmol) in 10 mL of THF and conc. HCl (1 mL) was stirred atroom temperature for 48 h. The mixture was concentrated under reducedpressure. The pH was adjusted to 8 using sat. aq NaHCO₃. The mixture wasextracted with EtOAc, washed with brine, dried (Na₂SO₄) andconcentrated. The residue was triturated in EtOAc to give(3R,4R)-7-(3-chlorophenyl)-3-hydroxy-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(34 mg, 39% yield) as a white solid MS (ESI) calcd for C₂₁H₁₈ClN₅O₂:407.1. found: 408 [M+H].

Example 34. Preparation of(3R,4R)—N-(4,5-dimethylthiazol-2-yl)-3-hydroxy-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(3R,4R)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol

A mixture of(3R,4R)-7-chloro-3-((trimethylsilyl)oxy)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(1 g, 3.53 mmol), ((3-trifluoromethyl)phenyl)boronic acid (1.34 g, 7.06mmol), CS₂CO₃ (3.44 g, 10.6 mmol), Pd(dppf)Cl₂ (300 mg, 0.35 mmol) indioxane/H₂O (30 mL, 10:1) was reacted under microwave at 130° C. for 2.5h. Then reaction mixture was poured into water, extracted with EtOAc,washed with water then brine, dried (Na₂SO₄), concentrated. The residuewas purified through silica gel chromatography (PE/EA=4:1) to give(3R,4R)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol(600 mg, 39% yield). MS (ESI) calcd for C₁₆H₁₄F₃N₃O: 321.1;

Step 2. Synthesis of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of TBSCl (338 mg, 2.24 mmol),(3R,4R)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-3-ol(600 mg, 1.87 mmol), triethyl amine (415 mg, 4.11 mmol) and DMAP (22 mg,0.20 mmol) was stirred for 48 h. Additional TBSCl and TEA were requiredto consume the starting material. The crude residue was purified bycolumn chromatography to give(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(90 mg, 73% yield). MS (ESI) calcd for C₂₂H₂₈F₃N₃OSi: 435.20;

Step 3. Synthesis of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-N-(4,5-dimethylthiazol-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of phenyl (4,5-dimethylthiazol-2-yl)carbamate (25 mg, 0.10mmol),(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(30 mg, 0.05 mmol) and DMAP (6 mg, 0.05 mmol) in 5 ml of MeCN wasstirred at 65° C. overnight. The crude reaction mixture was purified byprep. TLC to give(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-N-(4,5-dimethylthiazol-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(30 mg, quant.). MS (ESI) calcd for C₂₈H₃₄F₃N₅O₂SSi: 589.22;

Step 4. Synthesis of(3R,4R)—N-(4,5-dimethylthiazol-2-yl)-3-hydroxy-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(3R,4R)-3-((tert-butyldimethylsilyl)oxy)-N-(4,5-dimethylthiazol-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(30 mg, 0.051 mmol) in THF (2 mL) was added TBAF/THF (0.1 mL, 0.1 mmol).The mixture was stirred at room temperature overnight, poured into waterand extracted with EtOAc. The organic layer was washed with water thenbrine, dried over anhydrous Na₂SO₄, filtered and evaporated to dryness.The residue was purified by prep. TLC (EtOAc) to give(3R,4R)—N-(4,5-dimethylthiazol-2-yl)-3-hydroxy-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a white solid (12 mg, 50% yield). MS (ESI) calcd for C₂₂H₂₀F₃N₅O₂S:475.13. found: 476 [M+H].

Example 35: Preparation of(4S)—N-(4-(oxazol-5-yl)pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of 4-(oxazol-5-yl)pyridin-2-amine (500 mg, 3.10 mmol) inpyridine (7804, 9.65 mmol) and dichloromethane (10 mL), cooled to 0° C.,was added phenyl chloroformate (466 μL, 3.72 mmol) over 1.5 h. Thereaction was stirred at 0° C. for 2 h. Water (15 mL) was added slowly,and additional dichloromethane was added. The organic layer wasseparated, washed with saturated sodium carbonate (20 mL) and brine (20mL), dried with sodium sulfate, and all solvent removed in vacuo. Theresidue was suspended in 5:1 petroleum ether:ethyl acetate for 30 min,then the suspension filtered to give phenyl(4-(oxazol-5-yl)pyridin-2-yl)carbamate (547 mg, 1.94 mmol, 63% yield).

A solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(110 mg, 0.361 mmol), phenyl (4-(oxazol-5-yl)pyridin-2-yl)carbamate (203mg, 0.722 mmol) and 4-(dimethylamino)pyridine (53.0 mg, 0.434 mmol) inacetonitrile (5 mL) was stirred at 60° C. overnight. The mixture waspurified by preparative HPLC to give(4S)—N-(4-(oxazol-5-yl)pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(24.3 mg, 0.0493 mmol, 14% yield). MS (ESI) calcd for C₂₅H₁₉F₃N₆O₂:492.2. found: 493.2 [M+H].

Example 36: Synthesis of(4S)—N-(3,5-bis(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of phenyl (3,5-bis(oxazol-5-yl)phenyl)carbamate

A mixture of 3,5-bis(oxazol-5-yl)aniline (100 mg, 0.44 mmol), phenylchloroformate (76 mg, 0.48 mmol) and pyridine (0.20 mL) indichloromethane (15 mL) was stirred at room temperature for 2 h. Thesolvent was removed in vacuo, and the remaining material purified bypreparative TLC (1:1 petroleum ether:ethyl acetate) to give phenyl(3,5-bis(oxazol-5-yl)phenyl)carbamate (140 mg, 0.40 mmol, 92% yield). MS(ESI) calcd for C₁₉H₁₃N₃O₄: 347.1.

Step 2. Synthesis of(4S)—N-(3,5-bis(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A mixture of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(70 mg, 0.23 mmol), phenyl (3,5-bis(oxazol-5-yl)phenyl)carbamate (140mg, 0.40 mmol) and DMAP (56 mg, 0.46 mmol) in acetonitrile (2 mL) wasrefluxed overnight. The solvent was removed in vacuo, and the remainingresidue purified by preparative TLC (10:1 dichloromethane:methanol) togive(4S)—N-(3,5-bis(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(13.7 mg, 0.0245 mmol, 11% yield). MS (ESI) calcd for C₂₉H₂₁F₃N₆O₃:558.2. found: 559.0.

Example 37. Preparation of tert-butyl((1-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate

To a solution of triphosgene (214 mg, 0.721 mmol) in acetonitrile (5 mL)was added a solution of tert-butyl((1-(3-aminophenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate (417 mg, 1.44mmol) in acetonitrile (5 mL) and triethylamine (2 mL). The resultingsuspension was stirred at room temperature for 10 min, then(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(Compound #; 302 mg, 0.989 mmol) and 4-(dimethylamino)pyridine (122 mg,1.00 mmol) were added as solids. The reaction was stirred at 80° C. for15 min. The reaction was cooled to room temperature, methanol (2 mL) wasadded, and the reaction was poured into saturated sodium bicarbonate (50mL), and extracted with dichloromethane (3×50 mL). The combined organiclayers were dried with magnesium sulfate, the solvent removed in vacuo,and the remaining material purified by flash chromatography (0% to 8%methanol in dichloromethane) to give tert-butyl((1-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate(579 mg, 0.933 mmol, 94% yield). MS (ESI) calcd for C₃₁H₃₁F₃N₈O₃: 620.3.found: 621.0 [M+H].

Example 38. Preparation of(4S)—N-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

tert-butyl((1-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate(160 mg, 0.258 mmol) was dissolved in trifluoroacetic acid (1.6 mL). Thereaction was stirred at 50° C. for 10 min, then all solvent was removedin vacuo to give(4S)—N-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidetrifluoroacetic acid salt (164 mg, 0.258 mmol, 100% yield). MS (ESI)calcd for C₂₆H₂₃F₃N₈O: 520.2. found: 521.0 [M+H].

Example 39: Synthesis of(4S)—N-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1: Preparation of(4S)—N-(6-bromopyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of triphosgene (1.81 g, 6.10 mmol) in acetonitrile (25 mL)was added a solution of 6-bromopyridin-2-amine (2.26 g, 13.1 mmol) inacetonitrile (25 mL). Triethylamine (8.00 mL, 57.4 mmol) was added, andthe reaction stirred at 80° C. for 30 min.(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(1.04 g, 3.41 mmol) and 4-(dimethylamino)pyridine (410 mg, 3.36 mmol)were added as solids, and the reaction stirred at 80° C. for 1 h. Thereaction was cooled to room temperature, poured into water (30 mL), andextracted with dichloromethane (2×50 mL). The combined organic layerswere dried with magnesium sulfate, and all solvents removed in vacuo.The remaining residue was purified by flash chromatography (30% to 100%ethyl acetate in pentane) to give(4S)—N-(6-bromopyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(1.12 g, 2.22 mmol, 65% yield). MS (ESI) calcd for C₂₂H₁₇BrF₃N₅O: 503.1.found: 503.8 [M+H].

Step 2: Preparation of(4S)—N-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A microwave vial was charged with(4S)—N-(6-bromopyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(46.0 mg, 0.0912 mmol), tetrakis(triphenylphosphine)palladium (4.8 mg,0.0042 mmol), cesium fluoride (180 mg, 1.18 mmol),1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(60.0 mg, 0.288 mmol), DME (1.5 mL), and water (150 μL). The microwavevial was sealed, and heated in the microwave at 100° C. for 3 h. Theorganic layer was separated, and the aqueous layer extracted with 10:1ethyl acetate:methanol (2×2 mL). The combined organic layers were driedwith magnesium sulfate, the solvent removed in vacuo, and the remainingresidue dissolved in DMSO (4 mL), filtered, and the filtrate purified bypreparative HPLC to give(4S)—N-(6-(1-methyl-1H-pyrazol-5-yl)pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidetrifluoroacetic acid salt (28.4 mg, 0.0460 mmol, 50% yield). MS (ESI)calcd for C₂₆H₂₂F₃N₇O: 505.2. found: 506.0 [M+H].

Example 40: Synthesis of(4S)—N-(3-(piperazin-1-ylmethyl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A vial was charged with triphosgene (75.0 mg, 0.253 mmol), and this wasdissolved in acetonitrile (2.5 mL). A solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(150 mg, 0.491 mmol) in acetonitrile (2.5 mL) was added, followed bytriethylamine (0.50 mL, 3.59 mmol). The reaction was stirred at roomtemperature for 4 h, and tert-butyl4-(3-aminobenzyl)piperazine-1-carboxylate (480 mg, 1.65 mmol) was addedas a solid, followed by DMAP (360 mg, 2.95 mmol). The reaction wasstirred at 80° C. for 16 h, and all solvents were removed in vacuo. Theremaining material was dissolved in trifluoroacetic acid (5.0 mL), andthe solution stirred at 50° C. for 20 min. Excess trifluoroacetic acidwas removed in vacuo, the remaining material dissolved in DMSO, and theresulting solution purified by preparative HPLC to give(4S)—N-(3-(piperazin-1-ylmethyl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidetrifluoroacetate (89.1 mg, 0.140 mmol, 29% yield). MS (ESI) calcd forC₂₈H₂₉F₃N₆O: 522.2.

Example 41: Synthesis of(4S)—N-(4-(piperazin-1-yl)pyrimidin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of tert-butyl4-(2-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)pyrimidin-4-yl)piperazine-1-carboxylate

To a solution of tert-butyl4-(2-aminopyrimidin-4-yl)piperazine-1-carboxylate (200 mg, 0.716 mmol)in THF (20 mL) at 0° C. was added sodium bis(trimethylsilyl)amidesolution (1.0 M in THF, 1.50 mL, 1.50 mmol). The reaction was warmed toroom temperature and stirred for 30 min, and a solution of (4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(327 mg, 1.07 mmol) in THF (5 mL) was added. The reaction was stirred atroom temperature for 1.5 h, then the solvent removed in vacuo and theremaining residue purified by prep TLC (3:1 petroleum ether:ethylacetate) to give tert-butyl4-(2-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)pyrimidin-4-yl)piperazine-1-carboxylate(160 mg, 0.262 mmol, 37% yield). MS (ESI) calcd for C₃₀H₃₃F₃N₈O₃: 610.3.

Step 2. Synthesis of(4S)—N-(4-(piperazin-1-yl)pyrimidin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

tert-butyl4-(2-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)pyrimidin-4-yl)piperazine-1-carboxylate(160 mg, 0.262 mmol) was dissolved in hydrochloric acid in ethyl acetate(2 M, 10 mL), and stirred for 30 min. All solvents were removed in vacuoto give(4S)—N-(4-(piperazin-1-yl)pyrimidin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(47 mg, 0.092 mmol, 35% yield). MS (ESI) calcd for C₂₅H₂₅F₃N₈O: 510.2.found: 511.0 [M+H].

Example 42: Synthesis of tert-butyl(2-(4-(3-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)carbamate

A vial was charged with triphosgene (180 mg, 0.607 mmol), and this wasdissolved in dichloromethane (3 mL). A solution of(9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(320 mg, 1.00 mmol) was added, and N,N-diisopropylethylamine (1.00 mL,5.74 mmol) was added. The reaction was stirred at room temperature for25 min, and then a solution of tert-butyl(2-(4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl)ethyl)carbamate (415 mg,1.37 mmol) in dichloromethane (5 mL) was added. The reaction was stirredat 40° C. for 16 h, then heated in a microwave at 120° C. for 1 h. Aftercooling, the solvent was removed in vacuo, and the remaining residuepurified by flash chromatography (0% to 8% methanol in dichloromethane)to give tert-butyl(2-(4-(3-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)carbamate(99.0 mg, 0.153 mmol, 15% yield). MS (ESI) calcd for C₃₃H₃₅F₃N₈O₃:648.3.

Example 43: Preparation of(4S)—N-(3-(2-(guanidinomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of 1H-pyrazole-1-carboximidamide hydrochloride (15 mg, 0.10mmol) and DIEA (17 μL, 0.10 mmol) in DMF (1 mL) was stirred at roomtemperature for 10 min, the(4S)—N-(3-(2-(aminomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(52 mg, 0.10 mmol) was added. The reaction mixture was stirred at roomtemperature overnight and then purified by prep-HPLC and lyophilized toobtain(4S)—N-(3-(2-(guanidinomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidetrifluoroacetic acid salt (25.5 mg, 38% yield). MS (ESI) calcd forC28H25F3N8O2: 562.21. found: 563 [M+H].

This general procedure was used to prepare other guanidines bysubstituting(4S)—N-(3-(2-(aminomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidewith the appropriate amine.

Example 44: Synthesis of(4S)—N-(pyrimidin-4-yl)-7-(3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(9S)-2-(3-(trifluoromethyl)piperidin-1-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

A 20 mL microwave vial was charged with a magnetic stir bar,(9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(629 mg, 3.00 mmol),[1,3-Bis(2,6-di-isopropylphenyl)-4,5-dihydroimidazol-2-ylidene]chloro]allylpalladium(II)(62.1 mg, 0.120 mmol), 3-trifluoromethylpiperidine (919 mg, 6.00 mmol)and potassium tert-butoxide (673 mg, 6.00 mmol). DME (7.0 mL) was added,the microwave vial capped, and heated to 90° C. for 2 h. After coolingto room temperature, methanol (5 mL) and silica gel (5 g) were added,and all solvents were removed in vacuo. The remaining silica gel slurrywas loaded atop a 40 g silica gel column, and flash chromatography (50%to 100% ethyl acetate in pentane) gave(9S)-2-(3-(trifluoromethyl)piperidin-1-yl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine2 as a 1:1 mixture of diastereomers (713 mg, 2.18 mmol, 73% yield). MS(ESI) calcd for C₁₆H₂₁F₃N₄: 326.2.

Step 2. Synthesis of(4S)—N-(pyrimidin-4-yl)-7-(3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)-7-(3-(trifluoromethyl)piperidin-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(40.0 mg, 0.128 mmol) in acetonitrile (2 mL) and pyridine (1 mL) wasadded triphosgene (26.0 mg, 0.0876 mmol) as a solid. The resulting redsolution was stirred at 50° C. for 1 h. 4-aminopyrimidine (95.0 mg, 1.00mmol) was then added as a solid, and the reaction stirred at 70° C. for6 h. After 6 h, most of the acetonitrile was removed under a nitrogenstream, and methanol (1 mL) and DMSO (2 mL) was added to the reaction.The resulting solution was purified by prep HPLC, and the isolatedmaterial lyophilized from acetonitrile/aqueous 1N HCl to give(4S)—N-(pyrimidin-4-yl)-7-(3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidehydrochloride (34.9 mg, 0.0743 mmol, 58% yield). MS (ESI) calcd forC₂₀H₂₂F₃N₇O: 433.2.

Example 45: Synthesis of(4S)-7-((S)-3-(dimethylamino)pyrrolidin-1-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(3S)—N,N-dimethyl-1-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)pyrrolidin-3-amine

A 20 mL microwave vial was charged with a magnetic stir bar,(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(978 mg, 5.00 mmol),[1,3-Bis(2,6-di-isopropylphenyl)-4,5-dihydroimidazol-2-ylidene]chloro]allylpalladium(II)(51.7 mg, 0.100 mmol), (S)—N,N-dimethylpyrrolidin-3-amine (1.14 g, 10.00mmol) and potassium tert-butoxide (1.12 mg, 10.00 mmol). DME (10.0 mL)was added, the microwave vial capped, and heated to 100° C. for 4 h.After cooling to room temperature, methanol (20 mL) and silica gel (5 g)were added, and all solvents were removed in vacuo. The remaining silicagel slurry was loaded atop a 40 g silica gel column, and flashchromatography (0% to 10% methanol in pentane) gave(3S)—N,N-dimethyl-1-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)pyrrolidin-3-amine(524 mg, 1.92 mmol, 38% yield). MS (ESI) calcd for C₁₅H₂₃N₅: 273.2.

Step 2. Synthesis of(4S)-7-((S)-3-(dimethylamino)pyrrolidin-1-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(3S)—N,N-dimethyl-1-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)pyrrolidin-3-amine(40.0 mg, 0.146 mmol) in acetonitrile (540 μL) and pyridine (150 μL) wasadded a solution of triphosgene (28.9 mg, 0.0975 mmol) in acetonitrile(310 μL). The reaction was stirred at 50° C. for 30 min, thentriethylamine (40 μL) was added. 3-aminopyridine (94 mg, 1.00 mmol) wasadded as a solid, and the reaction stirred at 60° C. for 16 h. After 16h, the reaction was cooled to room temperature, methanol (1 mL) wasadded, and the resulting solution purified by preparative HPLC. Theisolated material was lyophilized from acetonitrile/aqueous 1N HCl togive(4S)-7-((S)-3-(dimethylamino)pyrrolidin-1-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidehydrochloride (30.6 mg, 0.0712 mmol, 49% yield). MS (ESI) calcd forC₂₁H₂₇N₇O: 393.2.

Example 46: Synthesis of tert-butyl4-((9S)-10-(pyrimidin-4-ylcarbamoyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylateStep 1. Synthesis of tert-butyl4-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate

A 20 mL microwave vial was charged with a magnetic stir bar,(9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(500 mg, 2.38 mmol),[1,3-Bis(2,6-di-isopropylphenyl)-4,5-dihydroimidazol-2-ylidene]chloro]allylpalladium(II)(12.4 mg, 0.0240 mmol), tert-butyl homopiperazine-1-carboxylate (953 mg,4.76 mmol) and potassium tert-butoxide (534 mg, 4.76 mmol). DME (5.0 mL)was added, the vial was sealed, and heated in the microwave at 85° C.for 4 h. After cooling to room temperature, methanol (20 mL) and silicagel (5 g) were added, all solvents were removed in vacuo, and theremaining silica gel slurry was loaded atop a 40 g silica gel column.Flash chromatography (0% to 8% methanol in dichloromethane) gavetert-butyl4-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate(550 mg, 1.48 mmol, 62% yield). MS (ESI) calcd for C₂₀H₃₁N₅O₂: 373.2.found: 374.2 [M+H].

Step 2. Synthesis of tert-butyl4-((9S)-10-(pyrimidin-4-ylcarbamoyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate

A mixture of tert-butyl4-((9S)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate(500 mg, 1.34 mmol), pyrimidin-4-yl-carbamic acid phenyl ester (570 mg,2.65 mmol), 4-dimethylaminopyridine (190 mg, 1.56 mmol) in acetonitrile(30 mL) was stirred at 60° C. for 3.5 h. Solvent was removed underreduced pressure, the residue was dissolved in dichloromethane, washedwith water, brine, and the organic layer dried over anhydrous Na₂SO₄.All solvent was removed in vacuo, and the remaining residue purifiedthrough silica gel chromatography with dichloromethane:ethyl acetate(2:1), then purified through preparative thin layer chromatography with3% methanol in dichloromethane to give tert-butyl4-((9S)-10-(pyrimidin-4-ylcarbamoyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate(363 mg, 0.734 mmol, 55% yield). MS (ESI) calcd for C₂₅H₃₄N₈O₃: 494.3.found: 495.4 [M+H].

Example 47: Synthesis of(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

tert-butyl4-((9S)-10-(pyrimidin-4-ylcarbamoyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocin-2-yl)-1,4-diazepane-1-carboxylate(400 mg, 0.808 mmol) was dissolved in 1M HCl in MeOH (20 mL), and thereaction mixture was stirred at room temperature for 1.5 h. All solventwas removed in vacuo. Water (20 mL) and potassium carbonate (344 mg,2.42 mmol) were added, and the mixture was stirred at room temperaturefor 1 h. Extraction with dichloromethane (3×5 mL) and drying the solventin vacuo gave(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(300 mg, 0.761 mmol, 94% yield). MS (ESI) calcd for C₂₀H₂₆N₈O: 394.2.

Example 48: Synthesis of(9S)-2-(4-(methylsulfonyl)-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

To a solution of(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(60.0 mg, 0.152 mmol) in dichloromethane (3 mL) was added triethylamine(46.1 mg, 0.457 mmol) and methanesulfonyl chloride (18.1 mg, 0.152mmol). The mixture was stirred at 0° C. for 1 h. The solution was washedwith water, brine, and the organic layer dried over anhydrous Na₂SO₄.The remaining solution was purified through preparative thin layerchromatography with 3% methanol in dichloromethane to give(9S)-2-(4-(methylsulfonyl)-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(30.0 mg, 0.0635 mmol, 42% yield). MS (ESI) calcd for C₂₁H₂₈N₈O₃S:472.2.

Example 49: Synthesis of(9S)-2-(4-methyl-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

To a solution of(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(100 mg, 0.254 mmol) in methanol (3 mL) was added formaldehyde (37% inwater, 20 μL) and 10% palladium on carbon (10 mg). The reaction wasstirred under hydrogen atmosphere for 1 h. The solvent was removed invacuo, and the remaining residue purified by preparative thin layerchromatography with 3% methanol in dichloromethane to give(9S)-2-(4-methyl-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(22.2 mg, 0.0543 mmol, 21% yield). MS (ESI) calcd for C₂₁H₂₈N₈O: 408.2.

Example 50: Synthesis of(9S)-2-(4-isopropyl-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

To a solution of(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(50.0 mg, 0.127 mmol) in dichloromethane (3 mL) was added acetone (12.9mg, 0.254 mmol). The mixture was stirred for 30 min, and sodiumcyanoborohydride (20.1 mg, 0.0759 mmol) was added as a solid, and thereaction stirred at room temperature overnight. All solvent was removedin vacuo, and the remaining residue purified by preparative thin layerchromatography (100% ethyl acetate) to give(9S)-2-(4-isopropyl-1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(16.8 mg, 0.0385 mmol, 30% yield). MS (ESI) calcd for C₂₃H₃₂N₈O: 436.3.

Example 51: Synthesis of(9S)—N-(pyrimidin-4-yl)-2-(4-(2,2,2-trifluoroethyl)-1,4-diazepan-1-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

To a solution of(9S)-2-(1,4-diazepan-1-yl)-N-(pyrimidin-4-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(100 mg, 0.254 mmol) in DMF (3 mL) was added sodium carbonate (80.7 mg,0.761 mmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (163 mg,0.508 mmol). The reaction was stirred at room temperature overnight,then diluted with dichloromethane (10 mL), and washed with water, brine,and dried with sodium sulfate. All solvent was removed in vacuo, and theremaining residue purified by preparative thin layer chromatography with3% methanol in dichloromethane to give(9S)—N-(pyrimidin-4-yl)-2-(4-(2,2,2-trifluoroethyl)-1,4-diazepan-1-yl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide(19.5 mg, 0.0409 mmol, 16% yield). MS (ESI) calcd for C₂₂H₂₇F₃N₈O:476.2.

Example 52: Synthesis of(4S)-7-(4-(2,2,2-trifluoroethyl)-1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineStep 1. Synthesis of tert-butyl4-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)-1,4-diazepane-1-carboxylate

To a solution of(4S)-7-chloro-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(500 mg, 2.56 mmol), N-Boc homopiperazine (953 mg, 4.76 mmol), potassiumtert-butoxide (534 mg, 4.76 mmol) in DME (5 mL) was added[1,3-bis(2,6-di-isopropylphenyl)-4,5-dihydroimidazol-2-ylidene]chloro]allylpalladium(II)(12.4 mg, 0.0240 mmol). The mixture was heated to 90° C. for 3 h. Aftercooling, water was added, and the mixture extracted with ethyl acetate(3×15 mL). The combined organic layers were washed with brine, driedwith sodium sulfate, filtered, and concentrated. The remaining residuewas purified by silica gel chromatography (5:1 pentane:ethyl acetate) togive tert-butyl4-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)-1,4-diazepane-1-carboxylate(610 mg, 1.70 mmol, 66% yield). MS (ESI) calcd for C₁₉H₂₉N₅O₂: 359.2.

Step 2. Synthesis of(4S)-7-(1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

A mixture of tert-butyl4-((4S)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepin-7-yl)-1,4-diazepane-1-carboxylate(610 mg, 1.70 mmol) and HCl in EtOAc (5M, 10 mL) was stirred at roomtemperature for 30 min. The solvent was removed to give(4S)-7-(1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(418 mg, 1.61 mmol, 95% yield). MS (ESI) calcd for C₁₄H₂₁N₅: 259.2.

Step 3. Synthesis of(4S)-7-(4-(2,2,2-trifluoroethyl)-1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

To a solution of(4S)-7-(1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(418 mg, 1.61 mmol) in DMF (5 mL) was added2,2,2-trifluoroethyltrifluoromethanesulfonate (748 mg, 3.23 mmol) andpotassium carbonate (666 mg, 4.83 mmol). The reaction was stirred atroom temperature overnight. The reaction was diluted with water,extracted with ethyl acetate (3×10 mL), and the combined organic layerswashed with brine, dried with sodium sulfate, filtered, andconcentrated. The remaining residue was purified by silica gelchromatography (6:1 pentane:ethyl acetate) to give(4S)-7-(4-(2,2,2-trifluoroethyl)-1,4-diazepan-1-yl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(320 mg, 0.937 mmol, 58% yield). MS (ESI) calcd for C₁₆H₂₂F₃N₅: 341.2.

Example 53: Synthesis of(4S)—N-(3-(oxazol-5-yl)phenyl)-7-((R)-3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideand(4S)—N-(3-(oxazol-5-yl)phenyl)-7-((S)-3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(120 mg, 0.241 mmol) was loaded onto a 30×250 mm chiralcel OD-H column.Elution with 20:80 ethanol:heptanes first eluted(4S)—N-(3-(oxazol-5-yl)phenyl)-7-((R)-3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(41.3 mg, 0.0828 mmol, 34% yield), [α]_(D) ²⁵=+32 (c, 0.09, MeOH),followed by(4S)—N-(3-(oxazol-5-yl)phenyl)-7-((S)-3-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(44.8 mg, 0.0899 mmol, 37% yield) [α]_(D) ²⁵=+18.5 (c, 0.11, MeOH). MS(ESI) calcd for C₂₅H₂₅F₃N₆O₂: 498.2. found: 499.3 [M+H].

Example 54. Preparation of(5S)—N-(5-fluoropyridin-3-yl)-8-(3-(trifluoromethyl)phenyl)-4,5-dihydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine-6(3H)-carboxamide1,1-dioxide Step 1. Synthesis of (S)-tert-butyl(1-((2,6-dichloropyridin-3-yl)sulfonyl)pyrrolidin-3-yl)carbamate

To a solution of 2,6-dichloropyridine-3-sulfonyl chloride (Org. ProcessRes. Dev. 2009, 13, 875-879) (3.40 g, 13.8 mmol) in dichloromethane (10mL) was added (S)-tert-butyl pyrrolidin-3-ylcarbamate (2.82 g, 14.5mmol), followed by triethylamine (3.00 mL, 21.5 mmol). The reaction wasstirred at room temperature for 30 min, then poured into saturatedsodium bicarbonate, and extracted with dichloromethane. The organiclayer was washed with brine, dried with Na₂SO₄, and the solvent removedin vacuo, to give (S)-tert-butyl(1-((2,6-dichloropyridin-3-yl)sulfonyl)pyrrolidin-3-yl)carbamate (5.47g, 13.8 mmol, 100% yield).

Step 2. Synthesis of (55)-8-chloro-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine 1,1-dioxide

To a solution of (S)-tert-butyl(1-((2,6-dichloropyridin-3-yl)sulfonyl)pyrrolidin-3-yl)carbamate (5.47g, 13.8 mmol) in dichloromethane (30 mL) was added trifluoroacetic acid(10 mL). The reaction was stirred at room temperature overnight, and allsolvent removed in vacuo. The remaining residue was dissolved in DMF (30mL), and sodium carbonate (10.0 g, 94.3 mmol) was added. The reactionwas stirred at 90° C. for 2 h. The reaction was cooled to roomtemperature, poured into ice water, and the resulting solution wasfiltered, and the solids washed with water. The collected solid wasdried, and purified by silica gel chromatography to give(5S)-8-chloro-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (1.80 g, 6.93 mmol, 50% yield).

Step 3. Synthesis of(5S)-8-(3-(trifluoromethyl)phenyl)-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide

A mixture of(5S)-8-chloro-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (800 mg, 3.08 mmol), 3-trifluoromethylbenzeneboronic acid(1.17 g, 6.16 mmol), cesium carbonate (3.00 g, 9.21 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (370 mg,0.453 mmol) in 10:1 dioxane:water (45 mL) was stirred at 110° C.overnight. After cooling, the solvent was removed in vacuo, and theresidue partitioned between dichloromethane and water. The organic layerwas separated, washed with water, brine, dried with Na₂SO₄, and thesolvent removed in vacuo. The remaining residue was purified by silicagel chromatography to give(5S)-8-(3-(trifluoromethyl)phenyl)-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (1.00 g, 2.71 mmol, 88% yield).

Step 4. Synthesis of(5S)-8-((S)-3-fluoropyrrolidin-1-yl)-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide

(S)-3-fluoropyrrolidine hydrochloride (1.10 g, 8.76 mmol) was added to asolution of(5S)-8-chloro-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (800 mg, 3.08 mmol) and sodium carbonate (1.60 g, 15.1 mmol)in DMF (10 mL). The reaction was stirred at 90° C. for 6 h, then pouredonto crushed ice, stirred, and filtered. The collected solid was washedwith water, dried, and purified by silica gel chromatography to give(5S)-8-((S)-3-fluoropyrrolidin-1-yl)-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (680 mg, 2.18 mmol, 71% yield).

Step 5. Synthesis of(5S)—N-(5-fluoropyridin-3-yl)-8-(3-(trifluoromethyl)phenyl)-4,5-dihydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine-6(3H)-carboxamide1,1-dioxide

(5S)-8-(3-(trifluoromethyl)phenyl)-3,4,5,6-tetrahydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine1,1-dioxide (70.0 mg, 0.190 mmol) was dissolved in DMF (3 mL), andsodium hydride (54 mg, 60% in oil, 1.35 mmol) was added. The reactionwas stirred at room temperature for 2 h, and phenyl(5-fluoropyridin-3-yl)carbamate (176 mg, 0.768 mmol) was added. Thereaction was stirred at room temperature for 1 h, poured into water, andextracted with dichloromethane. The organic layer was concentrated invacuo, and the remaining residue purified by preparative thin-layerchromatography to give(5S)—N-(5-fluoropyridin-3-yl)-8-(3-(trifluoromethyl)phenyl)-4,5-dihydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine-6(3H)-carboxamide1,1-dioxide (24.0 mg, 0.0473 mmol, 25% yield). MS (ESI) calcd forC₂₂H₁₇F₄N₅O₃S: 507.1. found: 508.1 (M+H)⁺.

The following compound was made in an analogous manner:(5S)—N-(5-fluoropyridin-3-yl)-8-((S)-3-fluoropyrrolidin-1-yl)-4,5-dihydro-2,5-methanopyrido[2,3-g][1,2,6]thiadiazocine-6(3H)-carboxamide1,1-dioxide.

Example 55. Synthesis of(4S)—N-(3-(4-((6-aminohexanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

A solution of tert-butyl((1-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate(190 mg, 0.306 mmol) in trifluoroacetic acid (3.0 mL) was stirred atroom temperature for 1 h, then the excess trifluoroacetic acid removedin vacuo. The remaining residue was dissolved in DMF (5.0 mL) andtriethylamine (1.0 mL), and 2,5-dioxopyrrolidin-1-yl6-((tert-butoxycarbonyl)amino)hexanoate (250 mg, 0.761 mmol) was added.The reaction was stirred at 80° C. for 30 min, then cooled to 65° C. and4N HCl (4 mL) was added. The reaction was stirred at 65° C. for 2 h,then filtered, and the filtrate purified by preparative HPLC. Theisolated material was lyophilized from acetonitrile/1N HCl to give(4S)—N-(3-(4-((6-aminohexanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidehydrochloride (208 mg, 0.310 mmol, 100% yield). MS (ESI) calcd forC₃₂H₃₄F₃N₉O₂: 633.3. found: 634.3 (M+H)⁺.

Example 56. Synthesis of(4S)—N-(3-(4-((6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)—N-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(44.2 mg, 0.0850 mmol) in acetonitrile (1.3 mL) and triethylamine (0.13mL, 0.933 mmol) was added biotinamidohexanoic acid N-hydroxysuccinimideester (40.9 mg, 0.090 mmol). The reaction was stirred at 65° C. for 2 h,then DMF (1 mL) was added, and the resulting reaction mixture filtered,and the filtrate purified by preparative HPLC to give(4S)—N-(3-(4-((6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(43.6 mg, 0.0448 mmol, 53% yield).

Example 57. Synthesis of(4S)—N-(3-(4-((3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-yl)thioureido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)—N-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(44.2 mg, 0.0850 mmol) in acetonitrile (1.3 mL) and triethylamine (0.13mL, 0.933 mmol) was added fluorescein isothiocyanate isomer I (35.0 mg,0.090 mmol). The reaction was stirred at 65° C. for 2 h, then DMF (1 mL)was added, and the resulting reaction mixture filtered, and the filtratepurified by preparative HPLC to give(4S)—N-(3-(4-((3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-yl)thioureido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(51.0 mg, 0.0498 mmol, 58% yield).

Example 58. Synthesis of(4S)—N-(3-(4-((3-((Z)-2-((1-(difluoroboryl)-1H-pyrrol-2-yl)methylene)-2H-pyrrol-5-yl)propanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of(4S)—N-(3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(12.2 mg, 0.0234 mmol) in acetonitrile (0.35 mL) and triethylamine(0.035 mL, 0.251 mmol) was added4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid, succinimidyl ester (10 mg, 0.0257 mmol). The reaction was stirredat 60° C. for 30 min, then the reaction mixture purified by preparativeHPLC to give(4S)—N-(3-(4-((3-((Z)-2-((1-(difluoroboryl)-1H-pyrrol-2-yl)methylene)-2H-pyrrol-5-yl)propanamido)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(5.8 mg, 0.0064 mmol, 27% yield).

Example 59. Synthesis of(9S)—N-(4-(acetamidomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamide

To a solution of(9S)—N-(4-(aminomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidehydrochloride (108 mg, 0.214 mmol) in pyridine (3.0 mL) was added aceticanhydride (28.0 μL, 0.300 mmol). The reaction was stirred at roomtemperature for 4 h, and the reaction mixture purified by preparativeHPLC to give(9S)—N-(4-(acetamidomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidetrifluoroacetate (111 mg, 0.178 mmol, 83% yield). MS (ESI) calcd forC₂₇H₂₆F₃N₅O₂: 509.2. found: 510.2 (M+H)⁺.

Example 60. Preparation of(4S)—N-(3-(pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1: Synthesis of tert-butyl3-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]thiazepine-5-carboxamido)phenyl)pyrrolidine-1-carboxylate

(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(A7, 0.150 g, 0.490 mmol) was dissolved in 5 ml methylene chloride andtreated with DIEA (193 ul, 1.08 mmol). The mixture was stirred at roomtemperature for 30 minutes. Tert-butyl3-(3-aminophenyl)pyrrolidine-1-carboxylate (0.141 g, 0.539 mmol) wasthen added and the solution was stirred over night at room temperature.The reaction was diluted with 5 ml methylene chloride and washed with 10ml of a saturated solution of sodium hydrogen carbonate. Organics werecollected and concentrated to dryness. Purification by silica columnchromatography using 5-100% ethylacetate in pentane afforded tert-butyl3-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)pyrrolidine-1-carboxylate(0.077 g, 26%). MS (ESI) calcd for C₃₂H₃₄F₃N₅O₃: 593.3. found: 594[M+H].

Step 2: Synthesis of(4S)—N-(3-(pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(S)Tert-butyl3-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)pyrrolidine-1-carboxylate:(0.077 g, 0.130 mmol) was dissolved in 5 mL of 4 N HCl in 1,4-dioxaneand stirred under nitrogen for 3 hours at room temperature. All solventwas then removed under reduced pressure and the resulting solid wasdried over night under vacuum to give(4S)—N-(3-(pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.078 g, 100%). MS (ESI) calcd for C₂₇H₂₆F₃N₅O: 493.21. found: 494[M+H].

Example 61. Preparation of(4S)—N-(3-(1-(2-amino-2-oxoethyl)pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)—N-(3-(pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(B579, 0.029 g, 0.058 mmol) was dissolved in 2 ml methylene chloride andthen treated with DIEA (31 ul, 0.174 mmol). Chloroacetamide (0.006 g,0.064 mmol) was then added and the reaction was heated to 60° C. overnight. The reaction was then cooled to room temperature and diluted with5 ml methylene chloride and washed with 10 ml of a saturated solution ofsodium hydrogen carbonate. Organics were concentrated to dryness andpurified via reverse phase chromatography on C18 using a gradient of5-95% acetonitrile in water with 0.1% trifluroacetic acid as additive togive(4S)—N-(3-(1-(2-amino-2-oxoethyl)pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.008 g, 23%). MS (ESI) calcd for C₂₉H₂₉F₃N₆O₂: 550.2. found: 551[M+H].

Example 62: Preparation of(4S)—N-(3-(1-(2-amino-2-oxoethyl)pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

(4S)—N-(3-(pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(B579, 0.020 g, 0.038 mmol) was dissolved in 1.5 ml methylene chlorideand then treated with DIEA (13 ul, 0.076 mmol). Acetylchloride (0.005 g,0.042 mmol) was then added and the reaction was stirred at roomtemperature over night. The reaction was then diluted with 5 mlmethylene chloride and washed with 10 ml of a saturated solution ofsodium hydrogen carbonate. Organics were concentrated to dryness andpurified via reverse phase chromatography on C18 to give(4S)—N-(3-(1-(2-amino-2-oxoethyl)pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.023 g, 88%). MS (ESI) calcd for C₂₉H₂₈F₃N₅O₂: 535.2. found: 536[M+H].

This general acylation procedure was used to prepare(4S)—N-(3-(1-propionylpyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideand(4S)—N-(3-(1-(cyclopropanecarbonyl)pyrrolidin-3-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide.

Example 63. Preparation of benzyl((5-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)oxazol-2-yl)methyl)carbamate

(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(0.276 g, 0.902 mmol) in 4 mL methylene chloride was combined with DIEA(400 uL, 2.261 mmol) and triphosgene (0.200 g, 0.676 mmol). The mixturewas then heated to 65° C. at which point benzyl((5-(3-aminophenyl)oxazol-2-yl)methyl)carbamate (0.321 g, 0.992 mmol) in4 mL methylene chloride was added dropwise slowly. The mixture was thenheated at 65° C. for 5 hours. The reaction was then cooled to RT and 10mL methylene chloride was then added followed by wash with 50 mL of asaturated solution of sodium hydrogen carbonate. The organic layer wascollected and concentrated to dryness under reduced pressure. Residuewas then purified via silica gel chromatography to afford benzyl((5-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)oxazol-2-yl)methyl)carbamate(0.082 g, 14%). MS (ESI) calcd for C₃₅H₂₉F₃N₆O₄: 654.22. found: 655[M+H].

Example 64. Preparation of(4S)—N-(3-(2-(aminomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

Benzyl((5-(3-((4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5-carboxamido)phenyl)oxazol-2-yl)methyl)carbamate(0.040 g, 0.06 mmol) was dissolved in 10 mL ethylacetate and degassedthree times under vacuum. Approximately 5 mg of Palladium (10% on Carbondegaussa type) was then added and the reaction vessel was purged withnitrogen then fitted with a hydrogen balloon. Stirring was theninitiated and the mixture was stirred under hydrogen for 2 hours. Thereaction vessel was then evacuated and purged with nitrogen. The solidswere removed via filtration and the solvent was removed under reducedpressure to give(4S)—N-(3-(2-(aminomethyl)oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(0.032 g, 100%) that was used without purification. MS (ESI) calcd forC₂₇H₂₃F₃N₆O₂: 520.51. found: 521 [M+H].

Example 65. Preparation of(4S)—N-(5-phenyl-1,3,4-oxadiazol-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of (4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate

To a solution of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(1.5 g, 4.92 mmol) and pyridine (0.74 mL, 9.84 mmol) in DCM (15 mL) wasadded phenyl chloroformate (1.1 mL, 8.85 mmol) at 0° C. dropwise. Theresulting mixture was stirred at that temperature for 2 h, then washedwith saturated aqueous NaHCO₃, dried over anhydrous Na₂SO₄, concentratedto afford the crude phenyl carbamate (1.8 g) as a brown oil, which wasused in the next step without further purification.

Step 2. Synthesis of(4S)—N-(5-phenyl-1,3,4-oxadiazol-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a solution of amine 5-phenyl-1,3,4-oxadiazol-2-amine (30 mg, 0.18mmol) in dry THF (5 mL) was added NaH (18 mg, 0.75 mmol) in portions at0° C., the reaction mixture was stirred at room temperature for 30 min.(4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(157 mg, 0.37 mmol) was then added and the mixture was stirredovernight. The resulting mixture was quenched with MeOH (2 mL),concentrated in vacuo by evaporator. The residue was purified byprep-TLC to give2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(50 mg, yield 54.9%) as a white solid.

This general procedure using (4S)-phenyl7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylatecould be used to prepare a variety of carboxamides by using theappropriate aryl-amines in place of 5-phenyl-1,3,4-oxadiazol-2-amine.

Example 66. Preparation of(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carbothioamideStep 1. Synthesis of 5-(3-isothiocyanatophenyl)oxazole

To a solution of 3-(oxazol-5-yl)aniline (160 mg, 1.0 mmol) and TEA (0.4mL, 3.0 mmol) in dry THF (6 mL) was added a solution of thiophosgene(0.152 mL, 2.0 mmol) in dry THF (1.5 mL) dropwise over 10 min at 0° C.under Argon atmosphere. The reaction mixture was stirred at ambienttemperature for 30 min, TLC showed 3-(oxazol-5-yl)aniline was fullyconsumed. The solvent was removed by evaporator under reduced pressure,the residue was diluted with water (10 mL) and ethyl acetate (25 mL),the organic layer was separated. The aqueous phase was extracted withethyl acetate (3×30 mL), the combined organic layers were washed withsaturated aqueous NaHCO₃ followed by water and brine, dried overanhydrous sodium sulfate, filtered and concentrated to give5-(3-isothiocyanatophenyl)oxazole which was used for the next reactionwithout any further purification.

Step 2. Synthesis of(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carbothioamide

(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(244 mg, 0.8 mmol) was dissolved in dry DMF (5 mL) and NaH (128 mg, 60%,3.2 mmol) was added in portions at 0° C. under Argon atmosphere. Themixture was warmed to room temperature and stirred for 1 h. A solutionof 5-(3-isothiocyanatophenyl)oxazole prepared above in DMF (3 mL) wasadded dropwise and the reaction mixture was stirred at ambienttemperature overnight. The resulting mixture was then quenched withsaturated aqueous NH₄Cl, extracted with ethyl acetate (3×15 mL), washedwith water and brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude was purified through silica gel chromatographyusing DCM:MeOH=10:1 to give title compound(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carbothioamide(186.1 mg, yield 46%).

Example 67. Preparation of(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanobenzo[b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of (S)-dimethyl2-((5-bromo-2-nitrophenyl)amino)succinate

The mixture of 4-bromo-2-fluoro-1-nitrobenzene (15.0 g, 68 mmol),(S)-dimethyl 2-aminosuccinate hydrochloride (15 g, 75 mmol) and DIPEA(36 mL) in DMSO (127 mL) was stirred at 100° C. for 2 h. After coolingdown, water (200 mL) was added and the mixture was extracted with ethylacetate (3×300 mL). The combined organic layers were washed with waterand brine, dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography(petroleum ether/ethyl acetate=4/1) to give (S)-dimethyl2-((5-bromo-2-nitrophenyl)amino)succinate (12.4 g, yield 51%)

Step 2. Synthesis of (S)-methyl2-(7-bromo-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl)acetate (SundiaE655-523-23)

The mixture of (S)-dimethyl 2-((5-bromo-2-nitrophenyl)amino)succinate(12.4 g, 34.4 mmol), Fe (22 g, 392 mmol) and AcOH (1.2 mL) in i-PrOH(250 mL) and water (50 mL) was stirred at reflux for 2 h. After cooling,the solid was filtered and the filtrate was concentrated. The residuewas diluted with DCM (300 mL) and water (300 mL), the organic layer wasseparated, and the aqueous phase was extracted with DCM (3×300 mL). Thecombined organic layers were washed with water and brine, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel chromatography (petroleum ether/ethylacetate=4/1) to give (S)-methyl2-(7-bromo-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl)acetate (8.8 g, yield86%) ¹H NMR (DMSO-d₆, 400 MHz): δ 10.43 (s, 1H), 6.84 (s, 1H), 6.75-6.73(m, 1H), 6.66-6.63 (m, 1H), 6.32 (s, 1H), 4.19-4.16 (m, 1H), 3.60 (s,3H), 2.78-2.72 (m, 1H), 2.68-2.61 (m, 1H).

Step 3. Synthesis of(S)-2-(7-bromo-1,2,3,4-tetrahydroquinoxalin-2-yl)ethanol

To the solution of (S)-methyl2-(7-bromo-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl)acetate (4.4 g, 14.7mmol) in THF (30 mL) was added BH₃Me₂S (10 M, 10 mL) at 0° C. over 15min in a dropwise fashion. The reaction was heated to reflux overnight.After cooling down, the mixture was quenched with 6N HCl (10 mL) and theresulting mixture stirred at 50° C. for 2 h. The mixture was thenbasified using 2N NaOH and brought to pH-8. The mixture was extractedwith DCM (3×50 mL) and the combined organic layers were washed withwater and brine, dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography(DCM/MeOH=20/1) to give(S)-2-(7-bromo-1,2,3,4-tetrahydroquinoxalin-2-yl)ethanol (2.4 g, yield63%).

Step 4. Synthesis of(4S)-7-bromo-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine

DDQ (2.7 g, 11.7 mmol) was added to a solution of PPh₃ (3.0 g, 11.7mmol) in DCM (100 mL) at room temperature.(S)-2-(7-bromo-1,2,3,4-tetrahydroquinoxalin-2-yl)ethanol (2.0 g, 7.8mmol) was added. The mixture was stirred at room temperature for 2 h.After removing the solvent, the residue was purified by silica gelchromatography (DCM/MeOH=40/1) to give(4S)-7-bromo-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine (1.5g, yield 81%).

Step 5. Synthesis of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine

To the mixture of(4S)-7-bromo-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine (600mg, 2.5 mmol), (3-(trifluoromethyl)phenyl)boronic acid (950 mg, 5.0mmol), Cs₂CO₃ (2.4 g, 7.5 mmol) in dioxane (60 mL) and water (6 mL) wasadded Pd(dppf)Cl₂ (204 mg, 0.25 mmol) at room temperature under N₂atmosphere. The mixture was stirred at 110° C. overnight. After coolingdown, the solid was filtered and the filtrate was concentrated. Theresidue was diluted with DCM (30 mL) and water (30 mL), the organiclayer was separated and the aqueous phase was extracted with DCM (3×30mL). The combined organic layers were washed with water and brine, driedover anhydrous sodium sulfate, filtered and concentrated. The residuewas purified by silica gel chromatography (DCM/MeOH=20/1) to give(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine(700 mg, yield 95%).

Step 6. Synthesis of(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanobenzo[b][1,4]diazepine-5(2H)-carboxamide

The mixture of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine(50 mg, 0.16 mmol), TEA (0.1 mL) and triphosgene (40 mg, 0.13 mmol) inTHF (5 mL) was stirred at 60° C. for 2 h. 3-(oxazol-5-yl)aniline (38 mg,0.24 mmol) was added. The mixture was stirred at 60° C. overnight. Aftercooling down, the resulting mixture was concentrated, the residue waspurified by prep-TLC (DCM/MeOH=20/1) to give(4S)—N-(3-(oxazol-5-yl)phenyl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanobenzo[b][1,4]diazepine-5(2H)-carboxamide(26.1 mg, yield 33%).

Example 68. Preparation of(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanobenzo[b][1,4]diazepine-5(2H)-carboxamide

The mixture of(4S)-7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-methanobenzo[b][1,4]diazepine(50 mg, 0.16 mmol), DMAP (52 mg, 0.42 mmol) and phenylpyridin-2-ylcarbamate (89 mg, 0.42 mmol) in CH₃CN (2.5 mL) was refluxedovernight. After cooling down, the solvent was removed. The residue waspurified by prep-TLC (DCM/MeOH=20/1) to give(4S)—N-(pyridin-2-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-methanobenzo[b][1,4]diazepine-5(2H)-carboxamide(24.4 mg, yield 35%).

Example 69. Preparation of(4S)—N-(pyridin-3-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of(4S)-7-(3-(trifluoromethyl)cyclohexyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine

This moiety was made using the general Negishi coupling procedure aboveto give(4S)-7-(3-(trifluoromethyl)cyclohexyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepineas a 11:1 mixture of diastereomers (482 mg, 36%). MS (ESI) calcd forC₁₆H₂₀F₃N₃: 311.16. found: 312 [M+H].

Step 2. Synthesis of(4S)—N-(pyridin-3-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

These compounds were made using the triphosgene urea coupling procedureabove to give(4S)—N-(pyridin-3-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a 9:1 mixture of diastereomers (38 mg, 62%). MS (ESI) calcd forC₂₂H₂₄F₃N₅O: 431.19. found: 432 [M+H].

Example 70. Preparation of(4S)—N-(pyrimidin-4-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

This mixture of diastereomers was made using the following protocol.Carbonyldiimidazole (CDI, 21 mg, 0.13 mmol) was slurried in DCM (1.5mL), followed by addition of 4-aminopyrimidine (13 mg, 0.13 mmol). Toget everything into solution, dioxane was added (0.5 mL). The mixturewas allowed to stir at room temp for 1 h under nitrogen atmosphere.(4S)-7-(3-(trifluoromethyl)cyclohexyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(41 mg, 0.13 mmol) was added in DCM (1 mL), and the reaction was allowedto stir overnight, then more CDI was added (21 mg) and the reactionheated to reflux for 4 h. The reaction was monitored by LCMS and theintermediate (before addition of 4-aminopyrimidine) was the majorreaction component. The reaction was cooled to room temp, concentrated,then more 4-aminopyrimidine (25 mg) was added in 1 mL DMSO (for bettersolubility). The reaction was warmed to 60° C. overnight, then 100° C.in a sealed tube for a second night. More 4-aminopyrimidine (25 mg) wasadded and the reaction sealed and heated to 120° C. in microwave for 1h. DCM (10 mL) was added, then 1 N HCl (3 mL). This was extracted withDCM (3×15 mL). Combined organics were washed with brine, dried withNa₂SO₄, filtered and concentrated. The crude product was purified bysilica gel column chromatography (0-10% MeOH/DCM), then again by prepHPLC to give(4S)—N-(pyrimidin-4-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(4 mg, 7%). MS (ESI) calcd for C₂₁H₂₃F₃N₆O: 432.19. found: 433 [M+H].

Example 71. Preparation of(4S)—N-(4,5-dimethylthiazol-2-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

This mixture of diastereomers was made using the following protocol.

General Procedure for Carbamate Formation:

Phenyl chloroformate (2.09 g, 13.3 mmol, 1.05 equiv.) was added dropwiseover 1.5 h to a cooled solution of 4,5-dimethylthiazol-2-amine (1.63 g,12.7 mmol, 1.0 equiv.) and pyridine (3.01 g, 38.2 mmol, 3.0 equiv.) inDCM (16 mL). The reaction was stirred with continued cooling for 2 h.Water (15 mL) was added slowly over 30 min, and then the mixture wasdiluted with DCM. The layers were separated and the organic layer waswashed with saturated aq. Sodium carbonate (20 mL), then brine (20 mL).The organic layer was then dried over Na₂SO₄, then concentrated underreduced pressure. The residue was suspended in EA/PE (1:5) for 30 min,then filtered to afford the phenyl (4,5-dimethylthiazol-2-yl)carbamate(1.7 g, 54%).

General Procedure for Urea Coupling Via Carbamate:

A mixture of phenyl (4,5-dimethylthiazol-2-yl)carbamate (80 mg, 0.322mmol, 2.0 equiv.),(4S)-7-(3-(trifluoromethyl)cyclohexyl)-2,3,4,5-tetrahydro-1,4-methanopyrido[2,3-b][1,4]diazepine(75 mg, 0.161 mmol, 1.0 equiv.) and DMAP (24 mg, 0.193 mmol, 1.2 equiv.)in acetonitrile (4 mL) were stirred at 60° C. overnight. TLC and LC/MSwere used to monitor reaction progress. The mixture was purified by prepHPLC to give(4S)—N-(4,5-dimethylthiazol-2-yl)-7-(3-(trifluoromethyl)cyclohexyl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(14.5 mg, 12%). MS (ESI) calcd for C₂₂H₂₆F₃N₅OS: 465.18. found: 466[M+H].

Example 72. Preparation ofN-(pyridazin-3-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(Compound xx) Step 1: Diethyl 3-aminopent-2-enedioate

A 250 mL 3-necked flask was charged with 6.00 g (29.7 mmol) of1,3-acetonedicarboxylate diethyl ester, 4.70 g (59.4 mmol) of ammoniumbicarbonate, and 80 mL of ethanol. The reaction was stirred at ambienttemperature for 24 h, then it was concentrated in vacuo. The residue wastaken up in 100 mL of water and extracted with ethyl acetate (3×100 mL).The combined organic layers were back extracted with brine (1×200 mL),dried over Na₂SO₄, filtered, and concentrated to give 5 g (87%) of theproduct as a colorless oil. This was used in the next reaction withoutfurther purification.

Step 2: Diethyl 3-aminopentanedioate

A 250 mL 3-necked flask was charged with 5.00 g (24.8 mmol) of diethyl3-aminopent-2-enedioate, 40 mL of ethanol, 10 mL of glacial acetic acid,and 3.1 g (49.6 mmol) of NaBH₃CN. The reaction was stirred at ambienttemperature for 2 h, then the solvents were removed in vacuo. Theresidue was taken up in water and extracted with ethyl acetate (3×100mL). The combined ethyl acetate layers were back extracted with brine(1×200 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo togive 4 g (80%) of the product as a colorless oil. This was used in thenext reaction without further purification.

Step 3: Diethyl 3-((6-chloro-3-nitropyridin-2-yl)amino)pentanedioate

A 250 mL 3-necked flask was charged with 1.8 g (9.8 mmol) of2,6-dichloro-3-nitropyridine, 4.0 g (19.7 mmol) of crude diethyl3-aminopentanedioate, 3.2 g (39.0 mmol) of NaHCO₃, and 60 mL oftetrahydrofuran. The reaction was stirred at 40° C. for 24 h, then thesolvent was removed in vacuo. The residue was dissolved in 100 mL ofwater, then extracted with ethyl acetate (3×100 mL). The combinedorganic phases were back extracted with brine (1×200 mL), dried overNa₂SO₄, filtered, and concentrated in vacuo. The residue was purifiedvia silica gel chromatography, eluting with 20/1 (v/v) hexanes/ethylacetate to give 2.7 g (80%) of the product as a light yellow solid.

Step 4: Diethyl 3-((3-amino-6-chloropyridin-2-yl)amino)pentanedioate

A 250 mL 3-necked flask equipped with a thermometer and a magnetic stirbar was charged with 2.7 g (7.5 mmol) of diethyl3-((6-chloro-3-nitropyridin-2-yl)amino)pentanedioate, 2.1 g (37.5 mmol)of iron powder, 60 mL of 2-propanol, 20 mL of water, and 675 mg (11.0mmol) of acetic acid. The mixture was stirred at 100 C for 1 h,monitoring by HPLC for the disappearance of the starting nitro compound.After the reaction was complete, the solids were filtered and washedwith 2-propanol (3×50 mL), then the combined filtrate and washings wasconcentrated in vacuo. The residue was dissolved in 100 mL of water andextracted with dichloromethane (3×50 mL). The combined organic layerswere back extracted with brine (1×50 mL), dried over Na₂SO₄, filtered,and concentrated to dryness. The crude product was purified via silicagel chromatography, eluting with 4/1 (v/v) hexanes/ethyl acetate to give1.8 g (75%) of the product as a gray solid.

Step 5: Ethyl2-(7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-pyrido[2,3-b][1,4]diazepin-4-yl)acetate

A 100 mL 3-necked flask equipped with a thermometer and a refluxcondenser was charged with 1.8 g (5.4 mmol) of diethyl3-((3-amino-6-chloropyridin-2-yl)amino)pentanedioate, 20 mL of toluene,and 1.0 mL (13.4 mmol) of trifluoroacetic acid. The mixture was stirredat reflux for 5 h, and the reaction was monitored by HPLC for thedisappearance of starting material. After the reaction was complete, thesolvents were removed in vacuo, then the residue was purified via silicagel chromatography, eluting with 3/1 (v/v) hexanes/ethyl acetate to give1.1 g (70%) of the product as an off-white solid.

Step 6:2-(7-chloro-2,3,4,5-tetrahydro-1H-pyrido[2,3-b][1,4]diazepin-4-yl)ethanol

A 50 mL 3-necked flask equipped with a nitrogen inlet, a refluxcondenser, and a thermometer was charged with 1.0 g (3.5 mmol) of ethyl2-(7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-pyrido[2,3-b][1,4]diazepin-4-yl)acetate,530 mg (14.0 mmol) of LiAlH₄, and 10 mL of tetrahydrofuran. The reactionwas stirred under N₂ at 60° C. for 6 h, monitoring for the appearance ofproduct by HPLC. The ester was reduced rapidly, but the lactam requireda longer time for complete reduction. When the reaction was complete,the mixture was cooled with an ice bath, 530 μL of water was added whilekeeping the internal temperature below 5° C., then the mixture wasstirred for 15 min. Next, 530 μL of 15% (w/w) NaOH(aq.) was added whilekeeping the internal temperature below 5 C, then the mixture was stirredfor 15 min. To complete the workup, 1590 μL water was added, then themixture was stirred at ambient temperature for 30 min. The solids werefiltered, then the precipitate was washed with tetrahydrofuran (3×50mL). The filtrate was concentrated in vacuo, then the residue waspurified via silica gel chromatography, eluting with 2/1 hexanes/ethylacetate to give 520 mg (65%) of the product as a light yellow solid.

Step 7:7-chloro-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine

A 50 mL 3-necked flask was charged with 500 mg (2.2 mmol) of2-(7-chloro-2,3,4,5-tetrahydro-1H-pyrido[2,3-b][1,4]diazepin-4-yl)ethanol,and 10 mL of 40% (w/w) HBr(aq.). The mixture was stirred at reflux for18 h, then it was cooled to ambient temperature and neutralized withsaturated NaHCO_(3(aq.)). The aqueous mixture was extracted with ethylacetate (3×50 mL), then the combined organic layers were back extractedwith brine (1×50 mL), dried over Na₂SO₄, filtered, and concentrated invacuo. The residue was purified via silica gel chromatography, elutingwith 3/1 hexanes/ethyl acetate to give 320 mg (70%) of the product as anoff-white solid.

Step 8. Synthesis of7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine

A dioxane/water mixture (10 mL/1 mL) was degassed and7-chloro-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine (250mg, 1.196 mmol) was added, followed by 3-(trifluoromethyl)phenylboronicacid (454 mg, 2.392 mmol), Pd(dppf)Cl₂ (97 mg, 0.19 mmol), and Cs₂CO₃(1.16 g, 3.588 mmol). The mixture was stirred at 110° C. for 12 hours,then concentrated and purified by column chromatography (PE/EtOAc=4/1)to give7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine(200 mg, 48%). MS (ESI) calcd for C₁₇H₁₆F₃N₃: 319.13.

Step 9. Synthesis ofN-(pyridazin-3-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

The following general urea coupling procedure was used:

The carbamate of pyridazin-3-amine (53.9 mg, 0.25 mmol, 2.0 equiv.),7-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine(40 mg, 0.12 mmol, 1.0 equiv.), and DMAP (18.4 mg, 0.15 mmol, 1.2equiv.) in acetonitrile (5 mL) were stirred at 60° C. overnight.Reaction progress was monitored by TLC and LC/MS. The reaction mixturewas loaded directly onto prep. TLC using 100% EtOAc as eluent to giveN-(pyridazin-3-yl)-7-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideas a white solid (16.9 mg, 30.6%). MS (ESI) calcd for C₂₂H₁₉F₃N₆O:440.16. found: 440.9 [M+H].

This general urea coupling procedure could be used to prepare a varietyof 7-(3-(trifluoromethyl)phenyl)-, 7-(3-chlorophenyl)-,7-(5-chloropyridin-3-yl)-, and7-(5-fluoropyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamidesby substituting the appropriate amine moiety for pyridazin-3-amine.

Example 73. Preparation of7-(3-chlorophenyl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of tert-butyl7-chloro-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate

This moiety was made using the following protocol. A mixture of7-chloro-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine (3.0g, 14.31 mmol), (Boc)₂O (4.6 g, 21.05 mmol, 1.5 equiv.), and DMAP (3.49g, 28.62 mmol, 2.0 equiv.) in THF (5 mL) was stirred at 60° C. for 2 h.TLC and LC/MS were used to monitor reaction progress. Water (30 mL) wasadded and the mixture was extracted with DCM (3×15 mL). The organicswere concentrated and the residue was purified by column chromatographyto give tert-butyl7-chloro-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylateas a white solid (4.5 g, 92%). MS (ESI) calcd for C₁₅H₂₀ClN₃O₂: 309.12.

Step 2. Synthesis of tert-butyl7-(3-chlorophenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate:(Sundia Prop. 455)

This moiety was made using the following protocol. To a degassed mixtureof dioxane/water (20 mL/1 mL) was added tert-butyl7-chloro-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.5 g, 4.85 mmol), (3-chlorophenyl)boronic acid (1.51 g, 9.70 mmol),Pd(dppf)Cl₂ (0.396 g, 0.485 mmol), and Cs₂CO₃ (4.74 g, 14.56 mmol). Themixture was stirred at 110° C. for 12 h, then concentrated and purifiedby column chromatography (PE/EA=2/1) to give tert-butyl7-(3-chlorophenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.2 g, 89%). MS (ESI) calcd for C₂₁H₂₄ClN₃O₂: 385.16.

This general coupling procedure could be used to prepare a variety of7-(3-substituted phenyl orpyridyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepines bysubstituting the appropriate boronic acid or boronic ester moiety for(3-chlorophenyl)boronic acid.

Step 3. Synthesis of7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine:(Sundia Prop. 455)

This moiety was made using the following protocol. Tert-butyl7-(3-chlorophenyl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.2 g, 3.1 mmol) was dissolved in HCl/MeOH (1 M, 20 mL) and thereaction mixture was stirred at room temp for 1.5 h, then concentratedin vacuo. Water (20 mL) and K₂CO₃ (3 g) were added. The mixture wasstirred at room temp for 2 h, then extracted with DCM (3×15 mL). Theorganics were concentrated to give7-(3-chlorophenyl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine(800 mg, 90%). MS (ESI) calcd for C₁₆H₁₆ClN₃: 285.10.

Step 4. Synthesis of7-(3-chlorophenyl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

This compound was made using the general urea coupling procedure to give7-(3-chlorophenyl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(20.4 mg, 24%). MS (ESI) calcd for C₂₂H₂₀ClN₅O: 405.14. found: 406[M+H].

Example 74. Preparation of7-(5-fluoropyridin-3-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamideStep 1. Synthesis of tert-butyl7-(5-fluoropyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate

This moiety was made using the following protocol. To a degassed mixtureof dioxane/water (30 mL/3 mL) was added tert-butyl7-chloro-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.39 g, 4.5 mmol), (5-fluoropyridin-3-yl)boronic acid (1.27 g, 9.0mmol), Pd(dppf)Cl₂ (0.37 g, 0.45 mmol), and Cs₂CO₃ (4.40 g, 13.5 mmol).The mixture was stirred at 110° C. for 12 h, then concentrated andpurified by column chromatography (PE/EA=2/1) to give tert-butyl7-(5-fluoropyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.5 g, 89%). MS (ESI) calcd for C₂₀H₂₃FN₄O₂: 370.18.

Step 2. Synthesis of7-(5-fluoropyridin-3-yl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine

This moiety was made using the following protocol. TFA (20 mL) was addedto a solution of tert-butyl7-(5-fluoropyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxylate(1.50 g) in DCM (20 mL), and the reaction mixture was stirred at roomtemp for 3 h, then concentrated in vacuo. The residue was basified withsaturated NaHCO₃ solution and extracted with DCM (3×15 mL). The organicswere concentrated to give7-(5-fluoropyridin-3-yl)-2,3,4,5-tetrahydro-1,4-ethanopyrido[2,3-b][1,4]diazepine(1.2 g, 100%). MS (ESI) calcd for C₁₅H₁₅FN₄: 270.13.

Step 3. Synthesis of7-(5-fluoropyridin-3-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

This compound was made using the general urea coupling procedure to give7-(5-fluoropyridin-3-yl)-N-(pyridin-3-yl)-3,4-dihydro-1,4-ethanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(12.8 mg, 15%). MS (ESI) calcd for C₂₁H₁₉FN₆O: 390.16. found: 391 [M+H].

Example 75. Preparation of 3-bromo-5-(oxazol-5-yl)aniline Step 1.Synthesis of 5-(3-bromo-5-nitrophenyl)oxazole

To a solution of 3-bromo-5-nitrobenzaldehyde (1 g, 4.34 mmol) in DME (10mL) was added K₂CO₃ (1.2 g, 8.68 mmol), followed by1-((isocyanomethyl)sulfonyl)-4-methylbenzene (891 mg, 4.56 mmol). Thereaction mixture was stirred at reflux overnight. After cooling to roomtemp., EtOAc was added and the mixture was washed with H₂O twice thenwith brine. The organic layers were dried over MgSO₄, filtered, andconcentrated. Purification by silica gel chromatography (0% to 100%EtOAc in pentane gradient) afforded 5-(3-bromo-5-nitrophenyl)oxazole(762 mg, 65%) as an orange solid. MS (ESI) calcd for C₉H₅BrN₂O₃: 268.0,270.0.

Step 2. Synthesis of 3-bromo-5-(oxazol-5-yl)aniline

To a solution of 5-(3-bromo-5-nitrophenyl)oxazole (762 mg, 2.83 mmol) inTHF (14 mL) was added acetic acid (13.6 mL), followed by iron powder(474 mg, 8.49 mmol). The reaction mixture was stirred at 60° C.overnight. After cooling to room temperature, the mixture was pouredinto a saturated Na₂CO₃ solution (175 mL) and extracted with EtOAc (50mL×2). The combined organic layers were washed with brine, dried overMgSO₄, filtered, and concentrated to afford3-bromo-5-(oxazol-5-yl)aniline (697 mg) as a brown oil. This materialwas used without further purification. MS (ESI) calcd for C₉H₇BrN₂O:238.0, 240.0.

This general two-step procedure of oxazole formation followed by nitroreduction could be used to prepare 4-bromo-5-(oxazol-5-yl)aniline byusing 4-bromo-5-nitrobenzaldehyde.

Example 76. Preparation of 6-(oxazol-5-yl)pyridin-2-amine Step 1.Synthesis of 6-amino-N-methoxy-N-methylpicolinamide

To a slurry of 6-aminopicolinic acid (10.0 g, 72.5 mmol) in acetonitrile(150 mL) was added N,O-dimethylhydroxylamine hydrochloride (8.52 g, 87.0mmol), 1-hydroxybenzotriazole (11.8 g, 87.0 mmol),N-(3-dimethylamino)-N′-ethylcarbodiimide hydrochloride (16.7 g, 87.0mmol), and N,N-diisopropylethylamine (37.7 mL, 217 mmol). The mixturewas stirred at room temperature overnight, and the solvent removed invacuo. The residue was partitioned between 1N NaOH and ethyl acetate,and the aqueous layer was extracted three times with ethyl acetate. Thecombined organic layers were washed with brine, dried with sodiumsulfate, and the solvent removed in vacuo. The remaining residue waspurified by flash chromatography (ethyl acetate with 0.1% triethylamine)to give 6-amino-N-methoxy-N-methylpicolinamide (4.30 g, 23.7 mmol, 33%yield). MS (ESI) calcd for C₈H₁₁N₃O₂: 181.1.

Step 2. Synthesis of 6-(oxazol-5-yl)pyridin-2-amine

Lithium aluminum hydride (1.08 g, 28.5 mmol) was added to a solution of6-amino-N-methoxy-N-methylpicolinamide (4.30 g, 23.7 mmol) in THF (30mL). The reaction was stirred at room temperature for 90 min. Ethylacetate (30 mL) was added slowly, the reaction was filtered, and thefiltrate taken and all the solvent removed in vacuo to give6-aminopicolinaldehyde, which was taken on crude to the next step.

To a solution of the above aldehyde in methanol (20 mL) was addedp-toluenesulfonylmethyl isocyanide (13.9 g, 71.2 mmol) and potassiumcarbonate (19.4 g, 140 mmol). The reaction was stirred at reflux for 2h, then all solvent removed in vacuo. The residue was partitionedbetween ethyl acetate (150 mL) and water (70 mL). The organic layer waswashed with brine, dried with sodium sulfate, and the solvent removed invacuo. The remaining residue was purified by flash chromatography (10%methanol in dichloromethane) to give 6-(oxazol-5-yl)pyridin-2-amine(2.00 g, 12.4 mmol, 52% yield over two steps). MS (ESI) calcd forC₈H₇N₃O: 161.1.

The following compounds were prepared in an analogous manner:4-(oxazol-5-yl)pyridin-2-amine; 5-(oxazol-5-yl)pyridin-3-amine.

Example 77: Synthesis of 3,5-bis(oxazol-5-yl)aniline Step 1. Synthesisof N¹,N³-dimethoxy-N¹,N³-dimethyl-5-nitroisophthalamide

To a solution of 5-nitroisophthalic acid (5.00 g, 23.7 mmol) indichloromethane (100 mL) was added oxalyl chloride (5.00 mL, 59.1 mmol),and the solution cooled to 0° C. DMF (1.0 mL) was added dropwise over 30min. The mixture was warmed to room temperature and stirred for 4 h. Allsolvents were removed in vacuo.

To a mixture of N,O-dimethylhydroxylamine hydrochloride (4.6 g, 47.1mmol) and triethylamine (6.60 mL, 47.4 mmol) in dichloromethane (80 mL)was added a solution of the above acid chloride in dichloromethane (20mL) at 0° C. Once the reaction was complete, the reaction mixture wasconcentrated in vacuo. The residue was partitioned between 1N sodiumhydroxide and ethyl acetate, the organic layer separated, and theaqueous layer extracted with ethyl acetate. The combined organic layerswere dried with sodium sulfate, the solvents removed in vacuo, and theresidue purified by silica gel chromatography (1:1 petroleum ether:ethylacetate) to give N¹,N³-dimethoxy-N¹,N³-dimethyl-5-nitroisophthalamide(4.00 g, 13.5 mmol, 57% yield). MS (ESI) calcd for C₁₂H₁₅N₃O₆: 297.1.

Step 2. Synthesis of 5-nitroisophthalaldehyde

Lithium aluminum hydride (2.70 g, 71.1 mmol) was added to a stirredsolution of N¹,N³-dimethoxy-N¹,N³-dimethyl-5-nitroisophthalamide (5.00g, 16.9 mmol) in THF (150 mL) at −40° C. The reaction was stirred at−40° C. for 4 h. 10% sodium hydroxide solution (2.7 mL) was addedslowly, followed by water (2.7 mL). The resulting solid was filtered,and the filtrate concentrated in vacuo to give 5-nitroisophthalaldehyde(1.37 g, 7.65 mmol, 45% yield). MS (ESI) calcd for C₈H₅NO₄: 179.0.

Step 3. Synthesis of 5,5′-(5-nitro-1,3-phenylene)bis(oxazole)

1-Isocyanomethanesulfonyl-4-methyl-benzene (7.40 g, 37.8 mmol) andanhydrous potassium carbonate (5.20 g, 37.8 mmol) were added to asolution of 5-nitroisophthalaldehyde (1.37 g, 7.65 mmol) in methanol(100 mL). The reaction was refluxed under nitrogen for 2 h. Aftercooling, the solvent was removed in vacuo. The residue was partitionedbetween ethyl acetate (150 mL) and water (70 mL). The organic layer wasremoved, and the aqueous layer extracted with ethyl acetate (3×150 mL).The combined organic layers were washed with brine, dried with sodiumsulfate, and concentrated to give crude5,5′-(5-nitro-1,3-phenylene)bis(oxazole) (1.70 g, 6.61 mmol, 86% yield).MS (ESI) calcd for C₁₂H₇N₃O₄: 257.0.

Step 4. Synthesis of 3,5-bis(oxazol-5-yl)aniline

A mixture of 5,5′-(5-nitro-1,3-phenylene)bis(oxazole) (1.70 g, 6.61mmol) and palladium on carbon (200 mg) in ethyl acetate (50 mL) wasstirred under hydrogen for 4 h. The solid was filtered, and the filtrateconcentrated in vacuo. The remaining residue was purified by silica gelchromatography (4:1 petroleum ether:ethyl acetate) to give3,5-bis(oxazol-5-yl)aniline (1.30 g, 5.72 mmol, 87% yield). MS (ESI)calcd for C₁₂H₉N₃O₂: 227.1.

Example 78. Preparation of tert-butyl(2-(4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl)ethyl)carbamate

A 20 mL microwave vial was charged with tert-butyl(2-bromoethyl)carbamate (551 mg, 2.50 mmol), sodium azide (460 mg, 7.05mmol), and DMF (5 mL). The vial was sealed, and heated in the microwaveat 110° C. for 12 h. The reaction mixture was poured into water (8 mL),and extracted with ethyl acetate (2×10 mL). The combined organic layerswere dried with magnesium sulfate, and all solvents removed in vacuo togive crude tert-butyl (2-azidoethyl)carbamate.

The crude tert-butyl (2-azidoethyl)carbamate was dissolved in THF (5 mL)and triethylamine (1 mL). 3-ethynylaniline (350 mg, 2.99 mmol) wasadded, followed by copper (I) iodide (15.0 mg, 0.0788 mmol). Thereaction was stirred at 60° C. for 2 h, then all solvents removed invacuo, and the remaining residue purified by flash chromatography (50%to 100% ethyl acetate in pentane) to give tert-butyl(2-(4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl)ethyl)carbamate 415 mg, 1.37mmol, 55% yield over 2 steps.) MS (ESI) calcd for C₁₅H₂₁N₅O₂: 303.2.

Example 79. Preparation of tert-butyl((1-(3-aminophenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate

To a solution of 3-azidoaniline (Chem. Commun. 2004, 888) (1.34 g, 9.99mmol) in THF (9.0 mL) and triethylamine (1.0 mL) was added tert-butylprop-2-yn-1-ylcarbamate (1.55 g, 9.99 mmol) and copper (I) iodide (40mg, 0.210 mmol). The reaction was stirred at 60° C. for 1 h, then allsolvents removed in vacuo. The remaining residue was purified by flashchromatography (0% to 80% ethyl acetate in pentane) to give tert-butyl((1-(3-aminophenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate (Compound #;1.71 g, 5.91 mmol, 59% yield). MS (ESI) calcd for C₁₄H₁₉N₅O₂: 289.1.found: 290.1 [M+H].

Example 80: Preparation of tert-butyl((1-(3-aminophenyl)-1H-1,2,3-triazol-5-yl)methyl)carbamate

To a vial with 3-azidoaniline (1.33 g, 9.92 mmol), tert-butylprop-2-yn-1-ylcarbamate (1.55 g, 9.99 mmol), andpentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride(15.9 mg, 0.020 mmol) was added toluene (10 mL). The reaction wasstirred at 100° C. for 72 h, then the reaction cooled to roomtemperature. Dichloromethane (5 mL) was added to dissolve any solids,and the remaining solution purified by silica gel chromatography (50% to80% ethyl acetate in pentane) to give tert-butyl((1-(3-aminophenyl)-1H-1,2,3-triazol-5-yl)methyl)carbamate (970 mg, 3.35mmol, 34% yield). MS (ESI) calcd for C₁₄H₁₉N₅O₂: 289.2.

Example 81: Preparation of N⁴,N⁴-dimethylpyrimidine-2,4-diamine

4-chloro-2-aminopyrimidine (495 mg, 3.82 mmol) was dissolved in aqueousdimethylamine (33%) in a sealed tube, and the reaction stirred at 100°C. overnight. After cooling, the reaction was diluted with water, andextracted with dichloromethane. The organic layer was washed with waterand brine, and dried with sodium sulfate, and the solvents removed invacuo to give N⁴,N⁴-dimethylpyrimidine-2,4-diamine (400 mg, 2.89 mmol,76% yield). MS (ESI) calcd for C₆H₁₀N₄: 138.1.

Example 82: Preparation of tert-butyl4-(2-aminopyrimidin-4-yl)piperazine-1-carboxylate

THF (20 mL) was added to a mixture of 4-chloro-2-aminopyrimidine (500mg, 3.87 mmol) and N-Boc piperazine (7.21 g, 38.7 mmol). The reactionwas stirred at 70° C. overnight. After cooling, the solvent was removedin vacuo and the remaining residue purified by silica gel chromatography(1:1 petroleum ether:ethyl acetate) to give tert-butyl4-(2-aminopyrimidin-4-yl)piperazine-1-carboxylate (650 mg, 2.33 mmol,60% yield). MS (ESI) calcd for C₁₃H₂₁N₅O₂: 279.2.

The following compound was made in an analogous manner:4-(4-methylpiperazin-1-yl)pyrimidin-2-amine

Example 83. Preparation of tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate Step 1.Synthesis of 2-bromo-5-nitrobenzaldehyde

To a solution of 2-bromobenzaldehyde (10.0 g, 53.7 mmol) in H₂SO₄ (100mL) was added KNO₃ (5.43 g, 53.7 mmol) in portions over 1 h at 0° C. Themixture was stirred for 40 min and additional KNO₃ (0.72 g) was added.The reaction mixture was stirred at 0° C. for 3 h then poured into icewater. The resulting precipitate was collected by filtration, rinsedwith water and recrystallized from EtOAc/Pentane to give2-bromo-5-nitrobenzaldehyde (11.7 g, 94% yield) as a white solid. MS(ESI) calcd for C₇H₄BrNO₃: 228.9.

Step 2. Synthesis of 5-(2-bromo-5-nitrophenyl)oxazole

A mixture of 2-bromo-5-nitrobenzaldehyde (1.0 g, 4.33 mmol), K₂CO₃ (1.79g, 12.9 mmol) and TosMIC (2.12 g, 10.8 mmmol) in MeOH was heated at 60°C. for 1.5 h. The mixture was concentrated. Water was added and thesolid collected by filtration, rinsed with water, MeOH then petroleumether to give 5-(2-bromo-5-nitrophenyl)oxazole (750 mg, yield 65%). asgrey solid. MS (ESI) calcd for C₉H₅BrN₂O₃: 228.9.

Step 3. Synthesis of tert-butyl(3-(4-nitro-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate

To a solution of 5-(2-bromo-5-nitrophenyl)oxazole (300 mg, 1.11 mmol) inDME (20 mL) was added tert-butyl prop-2-yn-1-ylcarbamate (431 mg, 2.77mmol) under N₂. CuI (21 mg, 0.11 mmol) and Pd(dppf)Cl₂ (78 mg, 0.11mmol) were added followed by TEA (0.5 mL). The reaction mixture washeated at 80° C. for 4 h, cooled to room temperature, poured into water,and extracted with EtOAc (3×50 mL). The combined organic layers werewashed with brine, dried and concentrated. The residue was purified bysilica gel column chromatography (EtOAc:Pentane=1:5) to give tert-butyl(3-(4-nitro-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate (350 mg,yield 92%) as a yellow oil. MS (ESI) calcd for C₁₇H₁₇N₃O₅: 343.1.

Step 4. Synthesis of tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate

A suspension of tert-butyl(3-(4-nitro-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate (3.8 g, 11.1mmol) and Fe (4.96 g, 8.86 mmol) in sat.aq NH₄Cl/MeOH (V/V=1:3) washeated at 60° C. for 4.5 h. The mixture was cooled to room temperature,passed through a pad of celite and the filtrate was concentrated. Theresidue was dissolved in EtOAc, washed with water, dried (Na₂SO₄) andconcentrated. The crude residue was purified by silica gel columnchromatography to give tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate (1.51 g,yield 44%) as a yellow oil. MS (ESI) calcd for C₁₇H₁₉N₃O₃: 228.9.

tert-butyl (3-(3-amino-5-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamatewas prepared from 3-bromo-5-nitrobenzaldehyde in a similar manner tothat described for tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate.

Example 84. Preparation of tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)propyl)carbamate

A mixture of tert-butyl(3-(4-nitro-2-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate (3.0 g, 8.75mmol) and 10 wt % Pd/C (1.0 g) in MeOH (100 mL) was stirred under H₂atmosphere (50 psi) for 16 h. The mixture catalyst was removed byfiltration, and the filtrate concentrated. The residue was purified bysilica gel column chromatography to give tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)propyl)carbamate (940 mg, yield 35%)as a yellow oil. MS (ESI) calcd for C₁₇H₂₃N₃O₃: 228.9.

tert-butyl (3-(3-amino-5-(oxazol-5-yl)phenyl)propyl)carbamate wasprepared from tert-butyl(3-(3-nitro-5-(oxazol-5-yl)phenyl)prop-2-yn-1-yl)carbamate in a similarmanner to that described for tert-butyl(3-(4-amino-2-(oxazol-5-yl)phenyl)propyl)carbamate.

Example 85. Preparation of2-(3,3-difluoropyrrolidin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

A mixture of2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1 g,4.48 mmol), 3,3-difluoropyrrolidine hydrochloride (1.9 g, 13.4 mmol),and K₂CO₃ (3 g, 22.4 mmol) in NMP (13 mL) was stirred at 110° C.overnight. A second portion of 3,3-difluoropyrrolidine hydrochloride(0.5 g) was added and stirred overnight. The mixture was filtered,washed with H₂O, added 2N HCl to adjust pH to 1. The mixture was washedwith EtOAc to remove residual2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine. Theaqueous layer was adjusted to pH 13 with K₂CO₃ solution, then extractedwith EtOAc to afford2-(3,3-difluoropyrrolidin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine(220 mg, 17%). MS (ESI) calcd for C₁₅H₂₁BF₂N₂O₂: 310.2.

This general procedure could be used to prepare4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyridineby using 3-(trifluoromethyl)pyrrolidine hydrochloride.

Example 86. Preparation of(S)-2-(3-fluoropyrrolidin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

A mixture of2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (200mg), (S)-3-fluoropyrrolidine hydrochloride (350 mg), and Na₂CO₃ (480) inIPA (3.5 mL) was stirred at 91° C. for 17 hrs. The mixture was filteredand concentrated. 2N HCl was added to adjust pH to 1 and extracted withEtOAc. The aqueous layer was adjusted to pH 7 with Na₂CO₃ solution andthe water was removed with toluene. The residue was taken up in EtOAc,filtered, and concentrated to afford(S)-2-(3-fluoropyrrolidin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine(95 mg, 36%). MS (ESI) calcd for C₁₅H₂₂BFN₂O₂: 292.2.

This general procedure could be used to prepare(R)-2-(3-fluoropyrrolidin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridineby using (R)-3-fluoropyrrolidine hydrochloride.

Example 87. Preparation of3,3-difluoro-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidineStep 1. Synthesis of 1-(3-bromophenyl)-3,3-difluoropyrrolidine

A mixture of 1,3-dibromobenzene (1 g, 4.24 mmol),3,3-difluoropyrrolidine hydrochloride (669 mg, 4.66 mmol), Pd₂(dba)₃(134 mg, 0.233 mmol), Cs₂CO₃ (3.32 g, 10.2 mmol), and BINAP (264 mg,0.424 mmol) in toluene (20 mL) was heated to reflux under a N₂atmosphere. After stirring overnight, the mixture was cooled to roomtemp. and concentrated. The concentrate was suspended in EtOAc (50 mL),washed with water (20 mL×3) and brine (20 mL), dried over Na₂SO₃ andconcentrated. Purification by column chromatography (hexane/EtOAc=100/1)afforded 1-(3-bromophenyl)-3,3-difluoropyrrolidine (645 mg, >100%) as acolorless oil. MS (ESI) calcd for C₁₀H₁₀BrF₂N: 261.0.

Step 2. Synthesis of3,3-difluoro-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine

A suspension of 1-(3-bromophenyl)-3,3-difluoropyrrolidine (899 mg, 3.43mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (957mg, 3.77 mmol), Pd(dppf)Cl₂ (75 mg, 0.103 mmol), KOAc (1 g, 10.29 mmol)in dioxane (18 mL) was heated to 85° C. under a N₂ atmosphere. Afterstirring overnight, the suspension was cooled to room temp. andfiltered. The filtrate was concentrated and purified by columnchromatography (hexane, then hexane/EtOAc=100/1) to afford3,3-difluoro-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrrolidine(Compound A#; 873 mg, 82%) as a white solid. MS (ESI) calcd forC₁₆H₂₂BF₂NO₂: 309.2.

This general two-step procedure could be used to prepare1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-(trifluoromethyl)pyrrolidineby using 3-(trifluoromethyl)pyrrolidine hydrochloride.

Example 88. Preparation of2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneStep 1. Synthesis of 1-allyl-3-bromobenzene

In a 3-neck flask, metal Mg (1.78 g, 73.76 mmol) was immersed in dryether (20 mL) under N₂ atmosphere. One-third of the volume of1,3-dibromobenzene (15 g, 63.58 mmol) in dry ether (20 mL) was addedinto the mixture. 1,2-Dibromoethane (0.1 mL) was added to initiate thereaction. After the reflux was steady, the remaining amount of the1,3-dibromobenzene solution was added dropwise at a rate to maintainreflux. Upon completion of addition, the mixture was stirred at refluxfor 1 hr. Then a solution of allylbromide (7.87 g, 65.12 mmol) in dryether (20 mL) was added dropwise. Upon completion of addition, thesuspension was stirred at reflux for 1 hr. The reaction was quenchedwith sat. NH₄Cl (100 mL) and the mixture was separated. The aqueousphase was extracted with ether (20 mL×2). The combined organic phaseswere washed with water (70 mL×2) and brine (30 mL), dried over Na₂SO₄,and concentrated to afford 1-allyl-3-bromobenzene (146 g) as a colorlessoil. This material was used without further purification. MS (ESI) calcdfor C₉H₉Br: 196.0.

Step 2. Synthesis of 3-(3-bromophenyl)propane-1,2-diol

To a solution of 1-allyl-3-bromobenzene (A#; 1 g, 5.08 mmol) inCH₃CN/H₂O (20 mL, v/v=4/1) were added NMO (1.3 g, 11.16 mmol) andK₂OsO₄.2H₂O (187 mg, 0.508 mmol). The mixture was stirred at room temp.for 2 days. The CH₃CN was removed under reduced pressure and theconcentrate was diluted with EtOAc. The mixture was filtered throughCelite and the filtrate was concentrated to afford3-(3-bromophenyl)propane-1,2-diol (Compound A#). This material was usedwithout further purification. MS (ESI) calcd for C₉H₁₁BrO₂: 230.0.

Step 3. Synthesis of 4-(3-bromobenzyl)-2,2-dimethyl-1,3-dioxolane

To a solution of 3-(3-bromophenyl)propane-1,2-diol (A#; 1.17 g, 5.06mmol) in acetone (25 mL) were added 2,2-dimethoxypropane (1.8 mL, 15.18mmol) and PTSA (96 mg, 0.506 mmol). The mixture was stirred at roomtemp. overnight. The reaction mixture was concentrated and purified bycolumn chromatography (hexane/EtOAc=20/1) to afford4-(3-bromobenzyl)-2,2-dimethyl-1,3-dioxolane (Compound A#; 200 mg, 15%)as a pale yellow oil. MS (ESI) calcd for C₁₂H₁₅BrO₂: 270.0.

Step 4. Synthesis of2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of 4-(3-bromo-benzyl)-2,2-dimethyl-[1,3]dioxolane (A#; 800 mg,2.95 mmol) and bis(pinacolato)diboron (822 mg, 1.1 eq), Pd(dppf)Cl₂ (216mg, 0.1 eq) and KOAc (868 mg, 3.0 eq) in dioxane (15 mL) was degassedand heated to 85° C. under N₂. After stirring overnight at 85° C., theblack suspension was cooled to room temp. and filtered through Celite.The filtrate was concentrated and purified by column chromatography(hexane/EtOAc=40/1) to afford2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(Compound A#; 800 mg, 85%) as a colorless oil. MS (ESI) calcd forC₁₈H₂₇BO₄: 318.2.

Example 89. Preparation of(9S)—N-(4-(aminomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-meth-anopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidehydrochloride and2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-((6-oxo-6-((4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)benzyl)amino)hexyl)carbamoyl)benzoic acid

Step 1. Synthesis of (S)-Dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)glutarate

To a mixture of 2,6-dichloro-3-nitropyridine (40.0 g, 207 mmol),L-glutamic acid dimethyl ester hydrochloride (87.7 g, 414 mmol) andNaHCO₃ (69.6 g, 829 mmol) was added tetrahydrofuran (600 mL). Themixture was stirred at 40° C. for 24 h, while monitoring for thedisappearance of 2,6-dichloro-3-nitropyridine by HPLC. After thereaction was complete, the solids were filtered and washed with ethylacetate (3×100 mL). The combined filtrate and washings were concentratedin vacuo, and the residue was purified via silica gel chromatography(eluting with 10:1 (v/v) hexanes/ethyl acetate) to obtain (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)glutarate as a yellow solid. (60g, 87%). LRMS (m/z) 332.1 [M+H]⁺; HRMS (m/z): [M+H]⁺ calcd forC₁₂H₁₅N₃O₆Cl, 332.0649. found, 332.0651.

Step 2. Synthesis of (S)-Methyl3-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propanoate

To a mixture of (S)-dimethyl2-((6-chloro-3-nitropyridin-2-yl)amino)pentanedioate (20 g, 60.2 mmol),and iron powder (16.8 g, 301 mmol) was added 2-propanol (375 mL) andwater (125 mL). To the stirred mixture was added acetic acid (5.5 g,90.3 mmol), and the reaction was stirred at reflux for 1 h whilemonitored for the disappearance of starting material by HPLC. After thereaction was complete, the solids were filtered and washed with2-propanol (3×50 mL). The combined filtrate and washings wereconcentrated to dryness, then the residue was concentrated in vacuo toobtain (S)-methyl3-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propanoateas a dark yellow solid which was used in the next step without furtherpurification (15 g, 81%). LRMS (m/z) 270.1 [M+H]⁺; HRMS (m/z): [M+H]⁺calcd for C₁₁H₁₃N₃O₃Cl, 270.0645. found, 270.0645.

Step 3. Synthesis of(S)-3-(6-Chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propan-1-ol

To a solution of AlCl₃ (17.78 g, 133.3 mmol) in tetrahydrofuran (260 mL)under N₂ was added 2M LiAlH₄ in THF (200 mL, 400 mmol), dropwise, at arate to control gas evolution. This gave a solution of alane (AlH₃) inTHF. In a separate flask, a solution of (S)-methyl3-(6-chloro-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propanoate(26.0 g, 96.4 mmol) in THF (460 mL) was prepared under N₂, then cooledwith a dry ice/acetone bath. To this was added the alane solution,dropwise with stirring, over 2 h. When the addition was complete, thecooling bath was removed, and the reaction was allowed to warm toambient temperature. After 1.5 h, LCMS analysis showed that the reactionwas complete, and a solution of NaOH (17.6 g) in water (65 mL) was addedslowly to control the evolution of H₂. The suspension was allowed tostir for 18 h, after which the solids were removed by filtration. Theprecipitate was washed with ethyl acetate, then the filtrate andwashings were concentrated in vacuo. The product was purified via silicagel chromatography (0 to 10% gradient of MeOH in CH₂Cl₂) to obtain(S)-3-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propan-1-olas a yellow-orange solid (15.21 g, 69%). LRMS (m/z) 228.1 [M+H]⁺; HRMS(m/z): [M+H]⁺ calcd for C₁₀H₁₅N₃OCl, 228.0904. found, 228.0903.

Step 4. Synthesis of(5R,9S)-2-Chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

To(S)-3-(6-chloro-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-3-yl)propan-1-ol(12 g, 52.7 mmol) was added 48% (w/w) HBr_((aq.)) (160 mL), and thereaction was stirred at 90° C. for 18 h while monitoring thedisappearance of the starting alcohol by HPLC. After the reaction wascomplete, it was cooled to ambient temperature, then 1.2 M aq. NaHCO₃was added until pH 8 was achieved. The mixture was extracted with ethylacetate (3×100 mL), then the organic phase was washed with brine (1×100mL), dried (Na₂SO₄), filtered, and concentrated to dryness. The residuewas purified by silica gel chromatography (eluting with 2:1 (v/v)hexanes/ethyl acetate) to obtain(5R,9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocineas a light yellow solid (6.0 g, 55%). LRMS (m/z) 210.1 [M+H]⁺; HRMS(m/z): [M+H]⁺ calcd for C₁₀H₁₃N₃Cl, 210.0798. found, 210.0800.

Step 5. Synthesis of(9S)-2-(3-(Trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine

A solution of(9S)-2-chloro-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine3.0 g, 15.4 mmol), (3-(trifluoromethyl)phenyl)boronic acid (4.4 g, 23.1mmol), palladium acetate (344 mg, 1.54 mmol),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (X-Phos, 476 mg,3.08 mmol), and cesium carbonate (15 g, 46.2 mmol) in a 10 to 1 (v/v)mixture of 1,4-dioxane and water (60 mL) was heated at 90° C. for 24hours. The reaction was then cooled to ambient temperature, and dilutedwith ethyl acetate (150 mL). The mixture was washed with sat. aq. NaHCO₃(200 mL×3), then the organic layers was dried (MgSO₄) and concentratedto dryness. The resulting residue was purified by silica gelchromatography (10-100% ethyl acetate gradient in pentane) to obtain(9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocineas a light yellow solid (3.7 g, 75%). LRMS (m/z) 320.2 [M+H]⁺; HRMS(m/z): [M+H]⁺ calcd for C₁₇H₁₇N₃F₃, 320.1375. found, 320.1375.

Step 6. Synthesis of tert-Butyl4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methano-pyrido[2,3-b][1,4]diazocine-10-carboxamido)benzylcarbamate

To a solution of(9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine(999 mg, 3.23 mmol) in CH₂Cl₂ (30 mL) was added N,N-diisopropylethylamine (1.7 mL, 9.78 mmol) and the reaction mixture was cooled to 0° C.with an ice bath. Triphosgene (482 mg, 1.63 mmol) was then added in foursmall portions. The ice bath was removed and the mixture was allowed towarm to room temperature. The reaction progress was monitored byremoving a 500 uL aliquot and combining with methanol to assay forconversion to methyl carbamate via formation of the intermediatechloroformate. If any starting material remained, an additional portionof triphosgene (200 mg) was added and the reaction mixture stirred forfive hours at room temperature. Next, tert-Butyl 4-aminobenzylcarbamate(800 mg, 3.60 mmol) was added in two equal portions (400 mg each) to theabove mixture and the resulting mixture was stirred at room temperaturefor two hours. Saturated aqueous NaHCO₃ (30 mL) was added and theorganic phase was then separated and concentrated to dryness underreduced pressure. The residue was purified by silica gel chromatography(15-100% ethyl acetate gradient in pentane) to obtain tert-butyl4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)benzyl-carbamateas a white solid. (761 mg, 42%). LRMS (m/z) 568.2 [M+H]⁺; HRMS (m/z):[M+H]⁺ calcd for C₃₀H₃₃N₅O₃F₃, 568.2536. found, 568.2538.

Step 7. Synthesis of(9S)—N-(4-(aminomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-meth-anopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidehydrochloride

tert-Butyl4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)benzylcarbamate(759 mg, 1.34 mmol) was dissolved in 4N HCl (10 mL) and 1,4-dioxane andstirred under nitrogen for 1 hour at ambient temperature. The solventswere removed under reduced pressure and the resulting solid was driedovernight under vacuum to obtain(9S)—N-(4-(aminomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidehydrochloride as a light tan solid (763 mg, 100%). LRMS (m/z) 468.1[M+H]⁺; HRMS (m/z): [M+H]⁺ calcd for C₂₅H₂₅N₅OF₃, 468.2011. found,468.2010.

Step 8. Synthesis of2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-((6-oxo-6-((4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)benzyl)amino)hexyl)carbamoyl)benzoic acid

(9S)—N-(4-(aminomethyl)phenyl)-2-(3-(trifluoro-methyl)phenyl)-8,9-dihydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10(7H)-carboxamidehydrochloride (39 mg, 0.10 mmol) was dissolved in acetonitrile (2 mL)and methanol (0.2 mL). N,N-diisopropylethylamine (32 μL, 0.20 mmol) wasthen added followed by 6-[fluorescein-5(6)-carboxamido]hexanoic acidN-hydroxysuccinimide ester (50 mg, 0.085 mmol). The mixture was stirredat room temperature overnight, then the product was isolated by reversedphase HPLC (5-95% acetonitrile gradient in water modified with 0.1% TFA)to obtain2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((6-oxo-6-((4-((9S)-2-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetra-hydro-6H-5,9-methanopyrido[2,3-b][1,4]diazocine-10-carboxamido)benzyl)amino)hexyl)carb-amoyl)benzoicacid as a brown solid (26 mg, 35%). LRMS (m/z) 939.2 [M+H]⁺; HRMS (m/z):[M+H]⁺ calcd for C₅₂H₄₆N₆O₈F₃, 939.3329. found, 939.3328.

Example 90. Mini-hSIRT1 Design and Characterization

Proton-deuteron exchange mass spectrometry (HDX-MS) was performed on thefull-length hSIRT1 protein to identify and characterize the keyfunctional regions of hSIRT1. The rate of H-D exchange is highlydependent on the dynamic properties of the protein, with faster exchangeoccurring at solvent exposed and/or flexible regions and slower exchangeoccurring at the more buried and/or structurally rigid regions (Hamuro,Y. et al. (2003) J biomol Techniques: JBT 14, 171). Consistent with theprevious study on hSIRT1(19-747) (Hubbard, B. P. et al. (2013) Science339, 1216), full-length hSIRT1 contains three major structured regions:the catalytic core region, residues 229-516 (referred to as hSIRT1cchereafter) (Jin, L. et al. (2009) J Biol Chem 284, 24394 and Frye, R. A.(2000) Biochem Biophys Res Commun 273, 793), the N-terminal region of190-230 immediately preceding the catalytic core and a remote region inthe C-terminus following the catalytic core around 640-670.

To probe the STAC binding site on hSIRT1, HDX-MS was performed in theabsence or presence of STAC 1. Addition of 1 reduces the H-D exchangerate around residues 190-230 in the N-terminal domain of hSIRT1,suggesting that this region is involved in STACbinding. Additionally,the ¹H, ¹⁵N HSQC spectrum of the ¹⁵N-labeled hSIRT1(180-230) is welldispersed suggesting that it forms an autonomously folded domain.Addition of 1 to ¹⁵N-labeled hSIRT1(180-230) results in significantchemical shift perturbations and further supports direct interaction of1 with this region, hereafter referred to as the STAC-binding domain(SBD). Addition of 1 to hSIRT1 in the presence of a p53-derived peptidesubstrate (Ac-p53(W5)) (Dai, H. et al. (2010) J Biol Chem 285, 32695)results in perturbation of the H-D exchange rates both around the SBDand at the presumed substrate binding site (residues 417-424) in thecatalytic core, indicating that STAC binding in the N-terminal domainand substrate binding within the catalytic core of hSIRT1 are coupled.This is consistent with a previous observation that STACs enhancesubstrate binding to hSIRT1, thereby increasing hSIRT1 catalyticefficiency (Milne, J. C. et al. (2007) Nature 450, 712).

In contrast to the SBD, the C-terminal structural element (641-665)identified by HDX-MS is separated from the catalytic core by about 150residues and is predicted to contain several β-strands, referred to hereas C-terminal β-strands/sheet (CBS), similar to the previously reportedmurine Essential for SIRT1 Activity (ESA) peptide (19). hSIRT1cc onlyshows about one eighth of the activity of the full-length enzyme usingdeacetylation assay conditions previously reported (Dai, H. et al.(2010) J Biol Chem 285, 32695). The CBS peptide restores the catalyticactivity of hSIRT1cc in trans, to 80% of that of full-length hSIRT1,with EC₅₀=59 nM, consistent with previous observations (Kang, H. et al.(2011) Mol Cell 44, 203 and Marmorstein, R. et al. (2012) J Biol Chem287, 2468). We also designed a minimal CBS fragment covering only theβ-stranded region (642-658), which behaves similarly to the parental CBSpeptide. Kinetic characterization reveals that the CBS peptide restoresactivity by lowering the KM values for both peptide substrate and NAD⁺of hSIRT1cc by 4-5-fold (see Table 1).

TABLE 1 Steady-State Kinetics of hSIRT1 catalytic core in the absence orpresence of CBS peptide.^(a) K_(M) (μM) Group k_(cat) (s⁻¹)Ac-p53(W5)^(b) NAD^(+c) hSIRT1(229-516) 0.29 ± 0.01 163 ± 8 988 ± 73hSIRT1(229-516) + CBS 0.54 ± 0.01   37 ± 2.5 180 ± 14 hSIRT1(229-516) +mini-CBS 0.54 ± 0.01  49 ± 3 207 ± 19 ^(a)Data from PNC1/GDH assay.^(b)NAD⁺ concentration fixed at 1 mM. ^(c)Ac-p53(W) concentration fixedat 500 μM.

Taken together, the above data suggests a tri-partite architecture for aminimally functional hSIRT1 that includes; 1) the central coreconstituting the basic catalytic machinery, 2) the N-terminal SBD thatmediates STAC binding and activation, and 3) the C-terminal CBS peptidewhich stabilizes the catalytic core resulting in more efficientdeacetylase activity. Based on this, we designed hSIRT1 constructsencompassing all three of the minimal structural elements covalentlybound, which we termed mini-hSIRT1s. The constructs span 183-505 or183-516, which are connected to the CBS peptide via a flexiblepoly-glycine/serine linker (GS, (GGGS)₂, or (GGGS)₃) (Sauer, R. T. andRobinson, C. R. (1998) Proceedings of Nat Academy of Sciences of USA 95,5929). The K_(M) and k_(cat) values are comparable between mini-hSIRT1constructs and the full-length enzyme, as are the IC₅₀ values for thenon-competitive hSIRT1 inhibitors EX-527 or nicotinamide (NAM)confirming functional fidelity of mini-hSIRT1s (see Table 2). Inaddition there is an excellent correlation between mini-hSIRT1 and thefull length enzyme with respect to STAC-mediated activation across abroad set of chemotypes. Removal of the SBD completely abolishesSTAC-mediated activation of mini-hSIRT1, confirming the criticalimportance of this domain for activation. In contrast, mini-hSIRT1lacking the CBS retains a significant level of STAC activationdemonstrating that the CBS enhances but, is not required forSTAC-mediated activation. Finally, the E230K mutation also attenuatesSTAC-mediated activation in mini-hSIRT1 as in the full-length enzyme(Hubbard, B. P. et al. (2013) Science 339, 1216). Collectively, theseobservations demonstrate that at half the molecular size, mini-hSIRT1 isa fully functional and activatable surrogate for full-length hSIRT1.

TABLE 2 Steady-state kinetics of mini-hSIRT1 constructs.^(a) K_(M) (μM)hSIRT1 k_(cat) (s⁻¹) Ac-p53(W5)^(b) NAD^(+c) hSIRT1 (1-747) 0.37 ± 0.01  3.7 ± 0.8^(d) 70 ± 6 hSIRT1 (183-516) 0.38 ± 0.01   42 ± 4.9 769 ± 97hSIRT1 0.44 ± 0.02   16 ± 2.2 82 ± 6 (183-516-(GGGS)₂-641-665) hSIRT10.54 ± 0.01   14 ± 1.1 78 ± 8 (183-516-(GS)-641-665) hSIRT1 0.54 ± 0.0213 ± 2 112 ± 10 (183-505-(GGGS)₂-641-665) hSIRT1 0.49 ± 0.01 13 ± 3 222± 25 (229-516-(GGGS)₂-641-665) hSIRT1 0.43 ± 0.02 11 ± 3 218 ± 34(229-505-(GGGS)₂-641-665) hSIRT1 0.54 ± 0.01   42 ± 4.1 170 ± 19(183-505-(GGGS)₂-641-665) (R446A) hSIRT1 0.21 ± 0.01 30 ± 3 375 ± 24(183-505-(GGGS)₂-641-665) (R446E) hSIRT1 0.30 ± 0.01 33 ± 2 423 ± 41(183-505-(GGGS)₂-641-665) (E230K, R446E) hSIRT1 0.65 ± 0.01 23.4 ± 1  273 ± 26 (183-505-(GGGS)₂-641-665) (P231G, P232G) ^(a)Data from PNC1/GDHassay. ^(b)NAD⁺ concentration fixed at 2 mM. ^(c)Ac-p53(W) concentrationfixed at 400 μM. ^(d)Value from (1)

Example 91. Structure of the Mini-hSIRT1/STAC Complex

While the x-ray crystallographic structure of the hSIRT1 catalytic corehas been reported (Zhao, X. et al. (2013) J Med Chem 56, 963), nostructure of the full-length enzyme exists to our knowledge. Structureof the full length hSIRT1 has been challenging, in part, due to theconformational flexibility of the extended N- and C-terminal domains.The mini-hSIRT1 constructs afforded us the opportunity to crystallize anequivalently functional surrogate of the full-length enzyme. Wesuccessfully crystallized mini-hSIRT1 (183-505-(GGGS)2-CBS) with STAC 1used in the HDX-MS experiments and determined the structure of thecomplex (mini-hSIRT1/1) at 3.1 Å by molecular replacement using a searchmodel based on the homolog model of SIRT3 (Jin, L. et al. (2009) J Biolchem 284, 24394). Mini-hSIRT1 is composed of a catalytic core thatassumes a Rossmann-fold large lobe and a zinc-binding small lobe commonto all sirtuins, an N-terminal three-helical bundle SBD and a C-terminalβ-hairpin CBS. Interestingly, a STAC-mediated dimer of mini-hSIRT1related by crystallographic symmetry was observed in the crystallattices. Size exclusion chromatography (SEC) confirms that mini-hSIRT1forms dimer in solution in the presence of STAC 1. However, no formationof mini-hSIRT1 dimer is observed for a similar STAC 7 of the samechemotype. Given this observation and the fact that the STACconcentration used for crystallization is much higher than that used inthe biochemical assay measuring activation, it would appear thatdimerization observed in the crystal structure is not a requirement forhSIRT1 activation by STACs.

The CBS mediates β-augmentation with the six-stranded β-sheet of theRossmann-fold lobe of the catalytic domain, in agreement with the HDX-MSresults of hSIRT1cc perturbation upon CBS binding. The CBS-mediatedβ-augmentation appears to stabilize the active site of the hSIRT1catalytic core which restores the K_(M) values observed for bothacetylated peptide and NAD⁺ substrates. The N-terminal SBD forms anindependently folded three-helical bundle with 1 binding to thehelix-turn-helix (H2-T-H3) motif within the SBD, consistent with theHDX-MS, NMR and enzyme kinetic results. The major mini-hSIRT1/1 bindingsite is a relatively shallow hydrophobic surface with an off-center,deeper hydrophobic pocket, that the CF₃ group of 1 occupies. This isconsistent with the observed structure activity relationship (SAR)developed across multiple STAC chemotypes indicating the requirement ofoverall flatness of the core scaffold maintained by an intramolecularhydrogen bond (Vu, et al. (2009) J Med Chem, 52, 1275). A remarkablesimilarity in terms of domain configuration is observed between themini-hSIRT1 structure and that of yeast Sir2 with both having anN-terminal helical bundle and the C-terminal β-augmentation by aβ-hairpin beyond the typical Rossmann-fold large lobe (Hsu, H. C. et al.(2013) Genes & Dev 27, 64). However, yeast Sir2 doesn't include the 150amino acid insertion observed in hSIRT1 and appears as a natural“mini-SIRT1” in yeast. The N-terminal domain in Sir2 appears to beimportant for the allosteric activation by another yeast protein Sir4(Hsu, H. C. et al. (2013) Genes & Dev 27, 64). Though the architectureof the Sir2 N-terminal domain differs from the hSIRT1 SBD, the twoappear to have functionally conserved roles in allosteric activation ofthe catalytic core.

Example 92. Site-Directed Mutagenesis of the STAC Binding Pocket

We used site-directed mutagenesis of full-length hSIRT1 to confirm thekey residues of the SBD that were identified by the mini-hSIRT1structures. The following point mutants of full-length hSIRT1 weregenerated probing three classes of residues: a) residues which appear todirectly interact with activators (T219A, I223A, N226A, and I227A), b)coupling modulator Glu²³⁰ (Hubbard, B. P. et al. (2013) Science 339,1216) (E230K, E230A, and E230Q), and c) SBD residues with no apparentrole in activator binding (Q222A and V224A). None of the mutantssignificantly impaired the basal catalytic activity using the Ac-p53(W5)substrate or affected inhibition by EX-527, a TFA-p53 peptide(Ac-RHK-K^(TFA)-L-Nle-F-NH₂), or nicotinamide (NAM) (see Tables 4 and5).

The general impact of the mutations on activation was assessed bycomparing the fold-activation of wild-type versus mutant full-lengthSIRT1 using a structurally diverse set of 246 STACs tested at a fixedconcentration of 25 μM. Additionally, we investigated the effect of themutations on STAC binding versus activation by monitoring shifts intheir EC₅₀ and maximum activation values respectively using a panel offive compounds (STACs 1, 6-9). T219A, I223A, and I227A all exhibit broadimpairment of activation with increases in EC₅₀ compared to wild-typehSIRT1, implicating impaired activator binding consistent with thestructures (see Table 6). Interestingly, I223A was the mostcompound-dependent mutant with STAC-mediated activation ranging fromattenuated to enhanced. STACs showing enhanced activation are enrichedfor structures containing an ortho-CF₃ substituted phenyl ring. In thecrystal structures, Ile²²³ lies directly beneath the activator and linesthe pocket into which the meta-CF₃ of 1 inserts. The cavity created bymutation of Ile²²³ to Ala would be expected to better accommodate anortho substituent versus a meta-substitution. This observation furthervalidates the key molecular interactions governing STAC binding andpoints to strategies for altering STAC interaction with the SBD.

TABLE 4 Steady-state substrate substrate kinetics for wild-type andmutant full-length hSIRT1. K_(M) (μM) hSIRT1 k_(cat) (s⁻¹)^(a)Ac-p53(W5)^(a) Ac-p53(W5)^(b) NAD^(+a) wild-type 0.37 ± 0.01  3.7 ±0.8^(c)  2.7 ± 0.4^(c) 70 ± 6  T219A 0.35 ± 0.01 2.7 ± 0.5 1.7 ± 0.2 45± 4  Q222A 0.34 ± 0.01 7.1 ± 2.3 4.8 ± 0.6 35 ± 6  I223A 0.33 ± 0.02 2.5± 0.3 4.2 ± 0.4 96 ± 14 V224A 0.31 ± 0.01 6.2 ± 0.7 4.2 ± 0.3 43 ± 4 N226A 0.34 ± 0.02 1.8 ± 0.7 2.5 ± 0.2 85 ± 9  I227A 0.36 ± 0.01 5.3 ±0.7 3.4 ± 0.4 43 ± 6  E230K 0.36 ± 0.01 7.0 ± 0.7 5.4 ± 0.2 70 ± 5 E230A 0.41 ± 0.01 6.2 ± 0.6 6.2 ± 0.5 57 ± 2  E230Q 0.38 ± 0.01 3.2 ±0.7 7.5 ± 0.9 99 ± 17 I223R 0.27 ± 0.01 3.5 ± 0.5 1.4 ± 0.2 91 ± 13^(a)Data from PNC1/GDH assay. ^(b)Data from OAcADPr assay. ^(c)Valuesfrom (1)

TABLE 5 Inhibition of wild-type and mutant full-length hSIRT1. IC₅₀(μM)^(a) TFA-p53 hSIRT1 EX-527 7-mer^(b) NAM wild-type 0.140 ± 0.0200.680 ± 0.070 92 ± 6 T219A 0.310 ± 0.040 0.490 ± 0.020 53 ± 4 Q222A0.110 ± 0.010 0.630 ± 0.050 53 ± 5 I223A 0.180 ± 0.020 0.540 ± 0.150 108± 10 V224A 0.140 ± 0.010 0.860 ± 0.080 64 ± 3 N226A 0.190 ± 0.010 0.350± 0.040 100 ± 9  I227A 0.220 ± 0.020 0.940 ± 0.060 92 ± 4 E230K 0.150 ±0.020 1.6 ± 0.3 54 ± 3 E230A 0.100 ± 0.030 1.3 ± 0.4 60 ± 3 E230Q 0.240± 0.020 0.860 ± 0.060 56 ± 3 I223R 0.210 ± 0.020 0.660 ± 0.060 87 ± 6^(a)Data from OAcADPr assay using the Ac-p53(W5) substrate. ^(b)TFA-p53peptide sequence: Ac-RHKK(TFA)L-Nle-F-NH₂.

TABLE 6 Substrate concentrations used in full-length hSIRT1 activationor inhibition assays. Activation assays Inhibition assays Ac-p53(W5)Ac-p53(W5) hSIRT1 (μM) NAD⁺ (μM) (μM) NAD⁺ (μM) wild-type 0.20 8.0 2 80T219A 0.20 4.5 2 45 Q222A 0.70 4.5 6.5 45 I223A 0.25 10 2.5 100 V224A0.60 4.5 6.5 45 N226A 0.20 8.0 2 80 I227A 0.40 4.5 4 45 E230K 0.40 8.0 480 E230A 0.40 8.0 4 80 E230Q 0.30 8.0 3 80 I223R 0.14 10 1.4 100

Asn²²⁶ appears to form a hydrogen bond between its carboxamide nitrogenand the carbonyl oxygen of 1 on the surface of the protein. However,activation of N226A was only minimally impaired compared to wild-type.The small contribution from this H-bond is likely due to its highsolvent exposure.

Mutation of Glu²³⁰ to either Lys or Ala has been recently reported tobroadly impair activation by STACs although the mechanism by which thisoccurs is unclear (Hubbard, B. P. et al. (2013) Science 339, 1216). Wetested activation of E230K, E230A, and E230Q full-length hSIRT1proteins. In all three Glu²³⁰ mutants, the maximum activation isimpaired without shifting the EC₅₀ (see Tables 7 and 8) suggesting arole for Glu²³⁰ in the formation or stabilization of the activatedconformation of hSIRT1. Activation of E230Q is also broadly impaired,indicating that the negative charge of Glu²³⁰ is important forstabilizing the activated conformation of hSIRT1 and that Glu²³⁰ likelyinteracts with a positively charged residue in the activatedconformation.

TABLE 7 Effect of hSIRT1 mutations on activator EC₅₀ values. compound: 16 7 8 9 EC₅₀ fold EC₅₀ fold EC₅₀ fold EC₅₀ fold EC₅₀ fold hSIRT1 (μM)shift^(a) (μM) shift^(a) (μM) shift^(a) (μM) shift^(a) (μM) shift^(a) WT0.30 1.00 0.77 1.00 2.20 1.00 0.48 1.00 0.77 1.00 T219A 1.00 3.30 2.383.10 8.83 4.01 3.96 8.26 4.12 5.36 Q222A 0.25 0.84 0.78 1.02 2.01 0.910.51 1.07 0.56 0.73 I223A 0.77 2.56 2.11 2.75 5.51 2.51 2.36 4.91 2.973.86 V224A 0.43 1.43 1.15 1.50 2.60 1.18 0.90 1.87 1.09 1.41 N226A 0.551.83 2.64 3.44 3.92 1.78 1.03 2.15 1.17 1.52 I227A 1.09 3.65 3.72 4.836.76 3.07 2.98 6.21 4.28 5.55 E230A 0.26 0.86 1.89 2.46 2.61 1.19 0.5301.11 1.23 1.60 E230K 0.15 0.50 2.92 3.81 3.21 1.46 0.70 1.46 1.63 2.12E230Q 0.37 1.23 2.15 2.80 3.36 1.53 0.58 1.21 1.34 1.74 ^(a)Fold shift =(mutant EC₅₀/wild-type EC₅₀) EC₅₀ values determined from activationdose-response curves using eq. 1. Activation was measured using theOAcADPr assay with Ac-p53(W5) substrate.

TABLE 8 Effect of hSIRT1 mutations on the maximum activation by STACs.compound: 1 6 7 8 9 fold fold fold fold fold hSIRT1 RV_(max) shift ^(a)RV_(max) shift ^(a) RV_(max) shift ^(a) RV_(max) shift ^(a) RV_(max)shift ^(a) WT 10.5 1.00 14.7 1.00 7.16 1.00 6.28 1.00 14.9 1.00 T219A5.76 1.99 14.3 1.03 3.25 2.73 4.22 1.64 10.4 1.47 Q222A 7.58 1.44 10.91.39 7.27 0.98 5.32 1.22 10.4 1.48 I223A 4.52 2.69 9.85 1.55 1.89 6.906.45 0.97 8.00 1.98 V224A 9.56 1.11 12.9 1.15 9.58 0.72 7.71 0.79 14.51.03 N226A 7.82 1.39 11.2 1.34 5.68 1.32 4.95 1.34 13.4 1.12 I227A 5.222.25 6.14 2.67 8.25 0.85 5.98 1.06 12.5 1.20 E230A 2.64 5.81 6.15 2.662.72 3.58 3.21 2.39 6.91 2.35 E230K 1.39 24.3 3.83 4.84 1.71 8.67 1.995.36 2.96 7.06 E230Q 2.28 7.40 4.67 3.73 2.57 3.93 2.72 3.07 5.14 3.35^(a) Fold shift = (wild-type RV_(max)−1)/(mutant RV_(max)−1) Maximumactivation values (RV_(max)) determined from activation dose-responsecurves using Eq. 1. Activation was measured using the OAcADPr assayassay with Ac-p53(W5) substrate.

In contrast to the above mutants, Q222A and V224A displayed normalactivation which is consistent with their positions away from the STACin the mini-hSIRT1/1 structure. Importantly, all of these data obtainedwith full-length hSIRT1 are consistent with what the mini-SIRT1 crystalstructures predict further validating the biochemical significance ofthese structures.

Despite the broad impact of the mutations described above, none of themcompletely abolished activation of hSIRT1 as seen with removal of theSBD. As Ile²²³ lies directly beneath the bound STAC and activation ofI223A is highly compound-dependent, we hypothesized that I223R hSIRT1would constitute the most highly activation-impaired full-length enzymeto date. Substitution from Ile to Arg introduces bulk and charge intothe hydrophobic STAC binding site which would be expected to disruptcompound binding based on the structure. I223R does not alter the basalcatalytic activity with either the Ac-p53(W5) or FOXO-3a substratepeptides or inhibition by EX-527, TFA-p53 peptide, or NAM (see Tables 4,5 and 9). However, activation is completely lost for all 246 activatorsfor both substrates.

TABLE 9 Steady-state kinetics for full-length hSIRT1 with FOXO-3a21-mer^(a). K_(M) peptide K_(M) NAD⁺ hSIRT1 k_(cat) (s⁻¹) (μM) (μM) wildtype 0.39 ± 0.02 50 ± 6 280 ± 40 I223R 0.25 ± 0.01 90 ± 6 460 ± 50^(a)Data from PNC1/GDH assay.

Example 93. Allosteric Coupling Between STAC and Substrate Binding

We investigated the mechanism of activation of hSIRT1 by STACs. To thatend, we determined a 2.73 Å structure of a quaternary complex ofmini-hSIRT1, 1, a 7 amino acid peptide substrate derived from p53(Ac-p53), and the non-hydrolyzable NAD⁺ analog carbaNAD and a 2.74 Åstructure mini-hSIRT1/1 in complex with a novel active-site directedinhibitor 2 that occupies the peptide and NAD⁺ binding sites. In thequaternary complex structure, the Ac-p53 peptide and carbaNAD bind tothe active site cleft between the large and small lobes. Ac-p53 adoptsan extended conformation, with main chain amide group forming hydrogenbonds with the residues Gly⁴¹⁵ and Glu⁴¹⁶ from the small lobe and theresidues Lys⁴⁴⁴ and Arg⁴⁴⁶ from the large lobe. The hydrogen bondsbetween the amide of the peptide+1 position and that of Arg⁴⁴⁶ render apotential interaction between the side chains for a hydrophobic+1residue, which may be important in STAC-mediated hSIRT1 activation (Dai,H. et al. (2010) J Biol Chem 285, 32695). The acetyllysine side chaininserts into a hydrophobic cavity lined by Phe⁴¹⁴, Leu⁴¹⁸ and Val⁴⁴⁵.The acetyl group is sandwiched between His³⁶³ and Phe²⁹⁷, with the ε-Nof the acetyllysine hydrogen bonded with the carbonyl oxygen of Val⁴¹²,which maintains the orientation and the extended conformation of theacetyl-lysine side chain. CarbaNAD also makes multi-point contacts withhSIRT1, most of which are similar to those observed in the ternarycomplex of hSIRT1 catalytic core/NAD/EX-527 analog (Zhao, X. et al.(2013) J Med Chem 56, 963). Some differences were noted, such as theamide group of the nicotinamide ring forms hydrogen bonds with Ile³⁴⁷and Asp³⁴⁸ in the C-pocket. In addition the 2′ and 3′ hydroxyl groups ofthe ribofuranose on the nicotinamide side form hydrogen bonds with thecarbonyl oxygen atom of acetyl-lysine, which helps to orient the C-1′atom of NAD for subsequent nucleophilic attack by the peptide N-ε-acetylgroup. Inhibitor 2 occupies both the acetyl-lysine binding site and thenicotinamide binding C-pocket of mini-hSIRT1, similar to the recentlyreported structure of the SIRT3/2 complex. Similar to SIRT3, binding ofsubstrates or the active-site inhibitor leads to domain closure,bringing the small and large lobes together (Szczepankiewicz, B. G. etal. (2012) J Org chem 77, 7319 and Jin, L. et al. J Biol Chem (2009)284, 24394). A more prominent conformational change upon active siteoccupancy is an upward movement of the N-terminal domain, which seems tohinge around Arg²³⁴ and brings the SBD closer to the active siteproviding a potential mechanism for the allosteric coupling of STACbinding and substrate binding sites through concerted motions. The hingeresidue, Arg²³⁴, is located within the polybasic linker (residues233-238) and anchors the N-terminal SBD to the catalytic core through asalt bridge formed between its guanidinium group and the carboxylategroup of Asp⁴⁷⁵ and hydrogen bonds to the carbonyl groups of His⁴⁷³ andVal⁴⁵⁹. Comparison of the SBDs in these three structures shows that thedomain is relatively rigid, with a superimposable STAC-bindinghelix-turn-helix (H2-T-H3) motif with only the first helix tilting outslightly in the mini-hSIRT1/1/2 complex structure. We assessed if theshort linker (230-233) between the SBD and the anchoring Arg²³⁴ might beimportant for the allosteric coupling by modulating the rigidity of thelinker through mutating Pro²³¹ and Pro²³² to Gly. Indeed, P231G/P232Gexhibits markedly attenuated STAC activation of mini-hSIRT1, supportingthe importance of this short linker to mediate the movement of the SBDand the consequential coupling of STAC binding and substrate binding.

HDX-MS data reveal that, in contrast to wild-type hSIRT1, STAC bindingto the E230K mutant no longer confers protection around the peptidebinding site in the E230K/1/Ac-p53(W5) complex. This indicates that theE230K mutation likely compromises the coupling between the STAC andsubstrate binding sites. To explore this further, a fluorescencepolarization (FP) assay was developed to measure STAC binding to hSIRT1and investigate the coupling effect in the presence of substrate. Afluorescein-linked STAC (3) which binds to full-length hSIRT1 with a IQof 0.3 μM was used as an FP probe and was effectively competed off byits parent compound 4. Binding of 3 was severely impaired for I223RhSIRT1 confirming the role of this residue shown in the mini-hSIRT1/1structures as being directly involved in STAC binding in the full-lengthenzyme. STACs were tested in the FP assay in the absence or presence ofAc-p53(W5) substrate exemplified by 5 which displayed enhanced bindingaffinity (K_(i)) in the presence of Ac-p53(W5) consistent with aK_(M)-lowering mechanism for activation. While the E230K mutant showsremarkably similar binding affinity for most STACs compared to thewild-type, enhancement of this binding by Ac-p53(W5) is absent orseverely attenuated. The HDX-MS and activation data together suggestthat Glu²³⁰ is not directly involved in STAC binding but is instead, acritical residue mediating the coupling of STAC and substrate binding topromote activation. This was further verified by determining thestructure of the E230K mini-hSIRT1 protein with compounds 1 and 2. Theoverall structures were similar to those of the wild-typemini-hSIRT1/1/2 ternary complexes, confirming that Glu²³⁰ does notdirectly participate in STAC binding but instead likely plays a dynamicrole during the allosteric coupling. Furthermore, a crystallographicdimer was observed with the E230K mini-hSIRT1/STAC structure, similar tothat seen for the wild-type protein structures, further indicating thatdimerization is unlikely to be required for the biochemical activationof hSIRT1 by STACs.

To assess the general requirement for a hydrophobic moiety on theacetyl-substrate for STAC-mediated SIRT1 activation (Dai, H. et al.(2010) J Biol Chem 285, 32695), we mutated the potential +1 Trpinteracting residue Arg⁴⁴⁶ to Ala based on the close proximity of Arg⁴⁴⁶to +1 position. R446A hSIRT1 has an increased K_(M) value for Ac-p53(W5)(see Table 2) as expected. However, it also shows attenuated activation,similar to what is observed for E230K/A mutants. Given this observation,the cationic nature of Arg⁴⁴⁶ and coupling between active site andSTAC-binding site, we made the mini-hSIRT1 R446E/E230K double mutant totest if Arg⁴⁴⁶ is a possible electrostatic partner for E230 with E230Kand R446E mini-hSIRT1 as controls. Whereas either E230K or R446E resultsin significant attenuation of STAC activation of mini-hSIRT1, theR446E/E230K double mutant partially restores STAC-mediated activation ofmini-hSIRT1 compared to E230K or R446E. These data support the existenceof a salt bridge between Glu²³⁰ and Arg⁴⁴⁶ in the activated conformationand are consistent with the proposed movement of the SBD to interactwith the catalytic core in the activated state.

All of mini-hSIRT1 constructs we discussed herein recapitulate three keyfeatures of full-length hSIRT1: 1) the steady state enzyme kinetics andinhibition, 2) the STAC activation profile across multiple chemotypes,3) and STAC activation impairment by E230K. We then used the mini-hSIRT1constructs to obtain the first reported structures of a fully functionalform of hSIRT1 with a bound STAC. The biochemical and structuralcharacterization reveals the hSIRT1 intra-molecular interactions betweenthe CBS and catalytic core, which are essential for the basaldeacetylation activity of hSIRT1. More importantly, the structures ofthe mini-hSIRT1/STAC complex reveal the detailed architecture of theSTAC binding site providing important information for futurestructure-based drug design. The comparison of the hSIRT1 structureswith different ligands bound suggests that the N-terminal SBD undergoesan upward movement along the conformational reaction coordinate,although the exact location of the SBD is likely influenced by crystalpacking. A potential mechanism for STAC activation can thus be inferred,namely the concerted motions of an upward movement of the SBD and domainclosure of the catalytic core to couple the STAC binding and thesubstrate binding. Biophysical characterization using FP andsite-directed mutagenesis using full-length hSIRT1 are fully consistentwith the structural observation of the mini-hSIRT1/STAC complex insupport of this model of STAC binding and activation. To our knowledge,the structures of the mini-hSIRT1/STAC complexes reported here representonly the second example of a synthetic allosteric activator bound to anenzyme besides glucokinase (Grimsby, J. et al. (2005) Science 301, 370).In summary, the results presented here provide unambiguous visual andfunctional proof of direct allosteric activation of hSIRT1 by smallmolecules with peptide substrates and provide a basis for furtherelucidation of the mechanism of hSIRT1 activation by STACs, andpotentially also by endogenous regulators of hSIRT1.

Example 94. Protein Cloning, Expression, and Purification

Mini-hSIRT1 constructs were cloned into a modified pET21b vector(Novagen). The protein was expressed in E. coli BL21-Gold (DE3) cells(Stratagene) as an N-terminal fusion to a hexahistidine affinity tagwith integrated TEV protease site. A single colony was inoculated in LBmedia containing 100 μg/ml ampicillin at 37° C., 250 rpm until the A600reached 0.3. The culture was then transferred to 16° C., 250 rpm untilthe A600 reached 0.6. Isopropyl 1-thio-β-D-galactopyranoside (IPTG) wasadded to a final concentration of 0.2 mM, and expression was continuedat 16° C., 250 rpm overnight. Cells were collected by centrifugation,and the pellet was resuspended in lysis buffer (25 mM HEPES, pH 7.5, 200mM NaCl, 5% glycerol, and 5 mM 2-mercaptoethanol) and sonicated to breakthe cells. Supernatant was separated from cell debris by centrifugationat 10,000×g for 40 min at 4° C. and loaded onto a Ni-NTA column (Qiagen)that equilibrated with the buffer containing 25 mM HEPES, pH 7.5, 200 mMNaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 20 mM imidazole. Thecolumn was washed with 5 column volumes of the buffer containing 25 mMHEPES, pH 7.5, 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 50mM imidazole, and eluted with the buffer containing 25 mM HEPES, pH 7.5,200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 250 mM imidazole.The eluted protein was dialyzed in lysis buffer and digested with TEVprotease (Invitrogen) to remove the N-terminal His tag at 4° C.overnight. The protein was loaded on a second Ni-NTA column equilibratedwith lysis buffer. The untagged protein was eluted by the buffercontaining 25 mM HEPES, pH 7.5, 200 mM NaCl, 5% glycerol, 5 mM2-mercaptoethanol, and 5 mM imidazole. The purified protein was dialyzedagainst the dialyzing buffer containing 20 mM Tris-HCl, pH 8.0, 250 mMNaCl, 5% glycerol, and 10 mM DTT, and concentrated. The protein wasfurther purified by a S200 column (GE Healthcare) to 95% purity asassessed by SDS-PAGE analysis stained by Coomassie Brilliant Blue R-250and concentrated to 10-15 mg/ml in the dialyzing buffer.

Human hsirt1 180-230 were cloned into a modified pET21b vector (Novagen)between BamHI and XhoI, which places expression under the control of theT7-lacO promoter. The protein was expressed in E. coli BL21-Gold (DE3)cells (Stratagene) as an N-terminal fusion to a hexahistidine affinitytag with integrated TEV protease site. A single colony was inoculated in100 ml LB media containing 100 ug/ml ampicillin at 37° C., 250 rpm forovernight. Then 20 ml LB media was inoculated to 1 L M9 media whichcontained ¹⁵NH₄Cl and Incubate them at 37° C. on an orbital shaker (200rpm) until OD600 was about 0.8. The culture was then transferred to 16°C., 250 rpm until the A600 reached 1.0. Isopropyl1-thio-β-D-galactopyranoside was added to a final concentration of 0.3mM, and expression was continued at 16° C., 200 rpm overnight. Cellswere collected by centrifugation, and the pellet was resuspended inlysis buffer (50 mM Hepes, 200 mM NaCl, 5% glycerol, 5 mM β-ME, pH 7.5)and sonicated to open the cells. Supernatant was separated from celldebris by centrifugation at 10,000×g for 40 min at 4° C. and loaded ontoa Ni-NTA column (Qiagen) that equilibrated with the buffer containing 50mM Hepes, 200 mM NaCl, 5% glycerol, 5 mM β-ME, pH 7.5. The column waswashed with 20 column volumes of the buffer containing 50 mM Hepes, 200mM NaCl, 5% glycerol, 5 m MB-ME and 20 mM imidazole, pH 7.5 and theneluted with the buffer containing 50 mM Hepes, 200 mM NaCl, 5% glycerol,5 mM β-ME and 250 mM imidazole, pH 7.5. The eluted protein was dialyzedin lysis buffer and digested with TEV protease (Invitrogen) to removethe N-terminal His tag at 4° C. overnight. The protein was loaded on asecond Ni-NTA column equilibrated with lysis buffer. The untaggedprotein was eluted by the buffer containing 50 mM Hepes, 200 mM NaCl, 5%glycerol, 5 mM β-ME and 10 mM imidazole, pH 7.5. The purified proteinwas concentrated and further purified by a S200 column (GE Healthcare)to get 95% purity as assessed by SDS-PAGE analysis stained by CoomassieBrilliant Blue R-250 and concentrated to 10 mg/ml in the 50 mM HEPES, 50mM NaCl, 0.5 mM TCEP, pH 6.5.

Example 95. Full-Length SIRT1 Production

Full-length human SIRT1 (hSIRT1) proteins were expressed with aC-terminal His₆ tag and purified as described in Hubbard. et al. (2013)Science 339, 1216, except for Q222A, and I223R SIRT1 which were purifiedusing an ÄKTAxpress™ (GE Lifesciences). Each cell paste was resuspendedin buffer A (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM imidazole, and0.1 mM TCEP) with 1,000 U Benzonase nuclease (Sigma Aldrich)supplemented with cOmplete, EDTA-free Protease Inhibitor CocktailTablets (Roche) on ice. Cells were disrupted by pulse sonication with50% on and 50% off for 12 minutes total at 40 W. Insoluble debris wasremoved by centrifugation. Clarified supernatant was directly loadedonto a 1 mL HisTrap FF Crude column (GE Lifesciences). After washingwith buffer A, SIRT1 was eluted with buffer B (50 mM Tris-HCl pH 7.5,250 mM NaCl, 500 mM imidazole and 0.1 mM TCEP). Protein was furtherpurified by size exclusion chromatography in buffer C (50 mM Tris-HCl pH7.5, 300 mM NaCl, 0.1 mM TCEP) using a Hi-load Superdex 200 16/60 column(GE Lifesciences). Enzyme concentrations were determined by Bradfordassay using BSA as a standard. Final protein purity was assessed by geldensitometry. Proteins were confirmed by LC/MS. All proteins weregreater than 90% pure except V224A and T219A (80%) and E230A (85%).

Example 96. SIRT1 Deacetylation Reactions

SIRT1 deacetylation reactions were performed in reaction buffer (50 mMHEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM DTT, and 1% DMSO) at 25° C.monitoring either nicotinamide production using the continuous PNC1/GDHcoupled assay (Smith, B. C. et al. (2009) Anal Biochem 394, 101) orO-acetyl ADP ribose (OAcADPr) production by mass spectrometry (Hubbard.et al. (2013) Science 339, 1216). Final concentrations of the PNC1/GDHcoupling system components used were 20 units/mL bovine GDH(Sigma-Aldrich), 1 uM yeast PNC1, 3.4 mM α-ketoglutarate, and 220 μMNADH or NADPH. An extinction coefficient of 6.22 mM⁻¹ cm⁻¹ and apathlength of 0.81 cm was used to convert the absorbance at 340 nm toproduct concentration for the 150 uL reactions used. Assays monitoringOAcADPr production were performed in reaction buffer with 0.05% BSA andtime points were taken by quenching the deacetylation reaction with astop solution which gave a final concentration of 1% formic acid and 5mM nicotinamide. Quenched reactions were diluted 5-fold with 1:1acetonitrile:methanol and spun at 5,000×g for 10 minutes to precipitateprotein before being analyzed with an Agilent RapidFire 200High-Throughput Mass Spectrometry System (Agilent, Wakefield, Mass.)coupled to an ABSciex API 4000 mass spectrometer fitted with anelectrospray ionization source. The p53-based Ac-p53(W5)(Ac-RHKK^(Ac)W-NH₂) and FOXO-3a 21-mer(Ac-SADDSPSQLSK^(Ac)WPGSPTSRSS-NH₂) peptides were obtained fromBiopeptide, Inc. Deacetylation assays used the Ac-p53(W5) substrateunless otherwise noted.

Substrate K_(M) determinations were performed by varying one substrateconcentration at a fixed, saturating concentration of the secondsubstrate. SIRT1 activation and inhibition assays were run in reactionbuffer with 0.05% BSA at 25° C. and analyzed using the OAcADPr assay.Enzyme and compound were pre-incubated for 20 minutes before addition ofsubstrates. For the activation screen of full-length hSIRT1, astructurally diverse set of 246 compounds was tested in duplicate at afinal concentration of 25 μM each. In order to be sensitive toK_(M)-modulating activators, substrate concentrations of approximatelyone-tenth their K_(M) values were used (see Table 5). Thedose-dependence of five compounds was tested and the fold-activationdata were described by Eq. 1

$\begin{matrix}{\frac{v_{x}}{v_{o}} = {b + \frac{{RX}_{{ma}\; x} - b}{1 + \frac{{EC}_{50}}{\lbrack X\rbrack_{o}}}}} & \left( {{Eq}{.1}} \right)\end{matrix}$where v_(x)/v₀ is the ratio of the reaction rate in the presence (v_(x))versus absence (v₀) of activator (X), RV_(max) is the relative velocityat infinite activator concentration, EC₅₀ is the concentration ofactivator required to produce one-half RV_(max) and b is the minimumvalue of v_(x)/v₀.

TABLE 5 Inhibition of wild-type and mutant full-length hSIRT1 IC₅₀(μM)^(a) TFA-p53 hSIRT1 EX-527 7-mer^(b) NAM wild-type 0.140 ± 0.0200.680 ± 0.070 92 ± 6 T219A 0.310 ± 0.040 0.490 ± 0.020 53 ± 4 Q222A0.110 ± 0.010 0.630 ± 0.050 53 ± 5 I223A 0.180 ± 0.020 0.540 ± 0.150 108± 10 V224A 0.140 ± 0.010 0.860 ± 0.080 64 ± 3 N226A 0.190 ± 0.010 0.350± 0.040 100 ± 9  I227A 0.220 ± 0.020 0.940 ± 0.060 92 ± 4 E230K 0.150 ±0.020 1.6 ± 0.3 54 ± 3 E230A 0.100 ± 0.030 1.3 ± 0.4 60 ± 3 E230Q 0.240± 0.020 0.860 ± 0.060 56 ± 3 I223R 0.210 ± 0.020 0.660 ± 0.060 87 ± 6^(a)Data from OAcADPr assay using the Ac-p53(W5) substrate. ^(b)TFA-p53peptide sequence: Ac-RHKK(TFA)L-Nle-F-NH₂.

Example 97. Protein Crystallization, Data Collection and StructureDetermination

The crystals of mini-hSIRT1/1 binary complex were obtained by hangingdrop vapor diffusion method at 18° C. The drop was composed of 1 μl ofprotein/compound mixture and 1 μl crystallization buffer of 0.2 MMagnesium chloride, 0.1 M Tris pH 8.5, and 16% w/v PEG 4000. Thecrystals of mini-hSIRT1/1/2 were obtained by hanging drop vapordiffusion method at 18° C. The drop was composed of 1 μl ofprotein/compound mixture and 1 μl crystallization buffer of 0.55 MSodium chloride, 0.1 M MES pH 6.5, and 20% w/v PEG 4000. The crystals ofmini-hSIRT1/1/p53-7mer/carbaNAD complex were obtained by hanging dropvapor diffusion method at 18° C. The drop was composed of 1 ul of theprotein/substrate mixture and 1 ul of the crystallization buffer of 5%v/v Tacsimate, pH 0.00.1 M HEPES pH 7.0 and 10% w/v PEG 5000 MME. Thecrystals of mini-hSIRT1(E230K)/1/2 were obtained by hanging drop vapordiffusion method at 18° C. The drop was composed of 1 μl ofprotein/compound mixture and 1 μl crystallization buffer of 0.2 MLithium Sulfate, 0.1 M Bis-Tris pH 6.5, 29% w/v PEG 3350.

The crystals were cryo-protected in mother liquor containing 20%glycerol before being flash-frozen in liquid nitrogen. Diffraction datawere collected at SSRF BL17U1, APS 21-ID-D or APS 21-ID-G beamlines andprocessed using the Xia2 program (Winter, G. (2010) J Appl Crystallogr43, 186). The molecular replacement software Phaser (McCoy, A. J. et al.(2007) J Appl Crystallogr 40, 658) was used to solve the structure witha search model containing residues 242-494 based on the homolog model ofSIRT3 (PDB code: 3GLU). Iterative structure refinement and modelbuilding were performed between Refmac5 (Murshudov, A. A. et al. (1997)Acta Crystallogr D Biol Crystallogr 53, 240) of the CCP4 package ((1994)Acta Crystallogr D Biol Crystallogr 50, 760) and Coot et al. (2004) ActaCrystallogr D Biol Crystallogr 60, 2126. Detailed information regardingthe diffraction data, refinement, and structure statistics is listed inTable 3.

TABLE 3 Data Processing and Refinement Statistics Mini-SIRT1Mini-SIRT1/1/p53 Mini-SIRT1/1 Mini-SIRT1/1/2 (E230K)/1/2 7-mer/CarbaNADData Collection Resolution (Å)* 45.67-3.10 39.98-2.73 40.17-3.2291.36-2.74 (2.81-2.74) (3.18-3.10) (2.81-2.73) (3.30-3.22) Space groupI2₁2₁2₁ P6322 P6322 I4122 Unit-cell parameters a (Å) 99.19 122.15 122.7294.51 b (Å) 111.64 122.15 122.72 94.51 c (Å) 132.52 104.92 105.88 356.84α (°) 90.00 90.00 90.00 90.00 β (°) 90.00 90.00 90.00 90.00 γ (°) 90.00120.00 120.00 90.00 Completemess (%)* 99.5 (99.8)  99.9 (100.0) 99.9(99.9)  99.5 (99.4) Redundancy* 4.8 (4.9) 17.6 (18.2) 17.4 (18.4)  9.6(9.9) Average I/σI* 17.4 (2.0)  38.8 (4.0)  38.1 (4.4)  20.7 (3.3)Rmerge (%)*  6.7 (78.1)  5.3 (80.3)  6.2 (80.6)  8.2 (82.9) RefinementResolution (Å)* 45.71-3.10 40.01-2.74 40.17-3.22 91.36-2.74 (2.81-2.74)(3.18-3.10) (2.81-2.73) (3.30-3.22) R_(work) (%)* 21.2 (33.9) 22.4(33.6) 22.8 (28.8)  18.3 (31.8) R_(free) (%)* 25.0 (37.6) 27.1 (43.8)25.1 (40.3)  23.5 (36.7) R.M.S.D in bond 0.005 0.007 0.006 0.010 lengths(Å) R.M.S.D in bond 1.089 1.14 1.079 1.538 angles (°) Mean B factors(Å2) 100.1 78.6 104.5 67.3 *Values in parentheses are for thehighest-resolution shell

Example 98. Nuclear Magnetic Resonance (NMR) Spectroscopy

The ¹H,¹⁵N HSQC NMR experiments were carried out at 25° C. on a BrukerAVANCE III 600 MHz NMR Spectrometer with cryoprobe using samplescontaining approximately 200 μM ¹⁵N-labeled SIRT1(180-230) in thepresence or absence of 400 μM 1. All NMR data were processed withNMRPipe (Delaglio, F. et al. (1995) J Biomol NMR 6, 277) and analyzedwith NMRView (Johnson, et al. (1994) J Biomol NMR 4, 603).

Example 99. Size Exclusion Chromatography (SEC) Assay

The assays were performed with a Superdex 75 10/300 GL column (GEhealthcare) injecting 100 μL samples containing 10 μM mini-hSIRT1 in theabsence or presence of 100 μM STAC, dissolved in 50 mM HEPES-NaOH, pH7.5, 150 mM NaCl, and 0.5 mM TCEP. Binding reactions were incubated for1 h at RT before injection into the column.

Example 100. Fluorescence Polarization (FP) Assay

FP experiments were carried out in 20 μL assay buffer (50 mM HEPES-NaOH,pH 7.5, 150 mM NaCl, and 1 mM DTT) at 25° C. The 384-well plates wereread on PHERAstar FS with excitation and emission wavelengths at 502 nmand 533 nm, respectively. For probe binding, increasing concentrationsof SIRT1 were added into 10 nM probe 3. The Binding isotherm wasdescribed by Eq. 2. For competitive binding mode, increasingconcentrations of competitors were added into the mixture of 10 nM 3 and0.3 μM SIRT1 wild-type or E230K mutant in the absence or presence or 15μM Ac-p53(W5). The competition data were described by Eq. 3. Theconversion of IC₅₀ to K_(i) was described by Eq. 4, where K_(d) is thebinding affinity of 3 to SIRT1, F₀ is the fraction of probe boundB/(B+F) and L₀ is the concentration of probe 3.

$\begin{matrix}{y = {\frac{x\left( {B - A} \right)}{x + K_{d}} + {Nx} + A}} & \left( {{Eq}{.2}} \right) \\{y = \frac{1}{1 + \frac{x}{{IC}_{50}}}} & \left( {{Eq}{.3}} \right) \\{K_{i} = {\frac{{IC}_{50}}{\frac{1}{1 - F_{0}} + \frac{L_{0}\left( {2 - F_{0}} \right)}{2K_{d}}} - {K_{d}\frac{F_{0}}{2 - F_{0}}}}} & \left( {{Eq}{.4}} \right)\end{matrix}$

Example 101. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

On-Exchange Experiment of SIRT1.

H/D-exchange reactions followed by pepsin digestion, desalting, HPLCseparation, and MS analysis were carried out using a fully automatedsystem, described in detail elsewhere (Hamuro, Y. et al. (2003) J BiomolTechniques: JBT 14, 171). Particular to this set of experiments,on-exchange reactions were initiated by mixing 20 μL of a SIRT1 stocksolution (0.77 mg/mL SIRT1, ±3.88 mM Ac-p53(W5), ±192 μM ligand, in 1.9%DMSO) and 20 μL of 100 mM phosphate, pH read 7.0 in D2O. The 50% D₂Omixture was incubated at 0° C. for 15, 50, 150, 500, 1,500, or 5,000 s.For SIRT1 (229-516), on-exchange reactions were initiated by mixing 4 μLof a SIRT1 stock solution (1.36 mg/mL SIRT1 (229-516), ±1.67 mMTrp-25mer) and 36 μL of 200 mM phosphate, pH read 7.0 in D2O. The 90%D₂O mixture was incubated at 0° C. for 15, 50, 150, 500, 1,500, or 5,000s. Addition of 20 μL of 1.6 M guanidine hydrochloride (GuHCl), 0.8%formic acid, pH 2.3, quenched the on-exchange reaction immediately priorto being analyzed.

Example 102. General Protein Process for Standard HDX Sample

The quenched solution was passed through a pepsin column (104 μL bedvolume) filled with porcine pepsin (Sigma, St Louis, Mo.) immobilized onPoros 20 AL media (Life Technologies, Carlsbad, Calif.) per themanufacturer's instructions, with 0.05% aqueous TFA (200 μL/min) for 2min. The digested fragments were temporarily collected onto a reversephase trap column (4 μL bed volume) and desalted. The peptide fragmentswere then eluted from the trap column and separated by a C18 column(BioBacis-18; Thermo Scientific, San Jose, Calif.) with a lineargradient of 13% solvent B to 40% solvent B over 23 min (solvent A, 0.05%TFA in water; solvent B, 95% acetonitrile, 5% buffer A; flow rate 10μL/min). Mass spectrometric analyses were carried out using a LTQOrbiTrap XL mass spectrometer (Thermo Fisher Scientific, San Jose,Calif.) with capillary temperature at 200° C.

Example 103. Digestion/Separation Optimization and Non-DeuteratedExperiment of SIRT1

Prior to H/D-exchange experiment, digestion and separation conditionswere optimized to yield high sequence coverage of SIRT1 by pepticfragments with high resolution under non-deuterated conditions. In thisstep, a mixture of 20 μL, of 0.77 mg/mL (9.2 μM) SIRT1 and 20 μL of H₂Owas quenched by the addition of 20 μL of various acidic buffers. ForSIRT1 (229-516), a mixture of 4 μL of a SIRT1 stock solution (1.36 mg/mLSIRT1 (229-516), ±1.67 mM Trp-25mer) and 36 μL of H₂O was quenched bythe addition of 20 μL of various acidic buffers. The quenched mixtureswere subjected to aforementioned general protein process. Thenon-deuterated peptic fragments were identified by Sequest in ProteomeDiscoverer 1.1 (Thermo Fisher Scientific, San Jose, Calif.).

Example 104. Fully Deuterated Experiment of SIRT1

The fully deuterated sample was prepared by incubating a mixture of 45μL of 0.77 mg/mL (9.2 μM) SIRT1 with 45 μL of 100 mM TCEP in D₂O, pH 2.5at 60° C. for 3 h. For SIRT1 (229-516), the fully deuterated sample wasprepared by incubating a mixture of 9 μL of 1.36 mg/mL (41.7 μM) SIRT1(229-516) with 81 μL of 100 mM TCEP in D₂O, pH 2.5 at 60° C. for 3 h.After incubation, the sample was kept at 0° C. before being quenchedidentically to an on-exchanged solution and subjected to the generalprotein process.

Example 105. Determination of Deuteration Level of Each Peptide afterOn-Exchange Reaction

The centroids of peptide isotopic envelopes were measured using thein-house-program developed in collaboration with Sierra Analytics(Modesto, Calif.). Corrections for back-exchange during the proteinprocessing step were made employing the following standard equation Eq.5 (Zhang, Z. et al. (1993) Protein Science 2, 522):

$\begin{matrix}{{{Deuteration}\mspace{14mu}{Level}\mspace{14mu}(\%)} = {\frac{{m(P)} - {m(N)}}{{m(F)} - {m(N)}} \times 100}} & \left( {{Eq}{.5}} \right)\end{matrix}$where m(P), m(N), and m(F) are the centroid value of partiallydeuterated (on-exchanged) peptide, non-deuterated peptide, and fullydeuterated peptide, respectively.

Example 106. Biological Activity

Mass spectrometry based assays were used to identify modulators of SIRT1activity. The TAMRA based assay utilized a peptide having 20 amino acidresidues as follows: Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE-NH₂(SEQ ID NO: 1), wherein K(Ac) is an acetylated lysine residue and Nle isa norleucine. The peptide was labeled with the fluorophore 5TMR(excitation 540 nm/emission 580 nm) at the C-terminus. The sequence ofthe peptide substrate was based on p53 with several modifications. Inaddition, the methionine residue naturally present in the sequence wasreplaced with the norleucine because the methionine may be susceptibleto oxidation during synthesis and purification. The Trp based assayutilized a peptide having an amino acid residues as follows:Ac-R-H-K-K(Ac)-W-NH2 (SEQ ID NO: 2).

The TAMRA based mass spectrometry assay was conducted as follows: 0.5 μMpeptide substrate and 120 μM βNAD⁺ was incubated with 10 nM SIRT1 for 25minutes at 25° C. in a reaction buffer (50 mM Tris-acetate pH 8, 137 mMNaCl, 2.7 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.05% BSA). The SIRT1 proteinwas obtained by cloning the SirT1 gene into a T7-promoter containingvector, which was then transformed and expressed in BL21(DE3) bacterialcells. Test compound was added at varying concentrations to thisreaction mixture and the resulting reactions were monitored. After the25 minute incubation with SIRT1, 10 μL of 10% formic acid was added tostop the reaction. The resulting reactions were sealed and frozen forlater mass spec analysis. Determination of the amount of deacetylatedsubstrate peptide formed (or, alternatively, the amount of0-acetyl-ADP-ribose (OAADPR) generated) by the sirtuin-mediatedNAD-dependent deacetylation reaction allowed for the precise measurementof relative SIRT1 activity in the presence of varying concentrations ofthe test compound versus control reactions lacking the test compound.

The Trp mass spectrometry assay was conducted as follows. 0.5 μM peptidesubstrate and 120 μM βNAD⁺ were incubated with 10 nM SIRT1 for 25minutes at 25° C. in a reaction buffer (50 mM HEPES pH 7.5, 1500 mMNaCl, 1 mM DTT, 0.05% BSA). The SIRT1 protein was obtained by cloningthe SirT1 gene into a T7-promoter containing vector, which was thenexpressed in BL21(DE3) bacterial cells and purified as described infurther detail below. Test compound was added at varying concentrationsto this reaction mixture and the resulting reactions were monitored.After the 25 minute incubation with SIRT1, 10 μL of 10% formic acid wasadded to stop the reaction. The resulting reactions were sealed andfrozen for later mass spec analysis. The relative SIRT1 activity wasthen determined by measuring the amount of 0-acetyl-ADP-ribose (OAADPR)formed (or, alternatively, the amount of deacetylated Trp peptidegenerated) by the NAD-dependent sirtuin deacetylation reaction in thepresence of varying concentrations of the test compound versus controlreactions lacking the test compound. The degree to which the test agentactivated deacetylation by SIRT1 was expressed as EC_(1.5) (i.e., theconcentration of compound required to increase SIRT1 activity by 50%over the control lacking test compound), and Percent Maximum Activation(i.e., the maximum activity relative to control (100%) obtained for thetest compound).

A control for inhibition of sirtuin activity was conducted by adding 1μL of 500 mM nicotinamide as a negative control at the start of thereaction (e.g., permits determination of maximum sirtuin inhibition). Acontrol for activation of sirtuin activity was conducted using 10 nM ofsirtuin protein, with 1 μL of DMSO in place of compound, to determinethe amount of deacetylation of the substrate at a given time pointwithin the linear range of the assay. This time point was the same asthat used for test compounds and, within the linear range, the endpointrepresents a change in velocity.

For the above assay, SIRT1 protein was expressed and purified asfollows. The SirT1 gene was cloned into a T7-promoter containing vectorand transformed into BL21(DE3). The protein was expressed by inductionwith 1 mM IPTG as an N-terminal His-tag fusion protein at 18° C.overnight and harvested at 30,000×g. Cells were lysed with lysozyme inlysis buffer (50 mM Tris-HCl, 2 mM Tris[2-carboxyethyl] phosphine(TCEP), 10 μM ZnCl₂, 200 mM NaCl) and further treated with sonicationfor 10 min for complete lysis. The protein was purified over a Ni-NTAcolumn (Amersham) and fractions containing pure protein were pooled,concentrated and run over a sizing column (Sephadex S200 26/60 global).The peak containing soluble protein was collected and run on anIon-exchange column (MonoQ). Gradient elution (200 mM-500 mM NaCl)yielded pure protein. This protein was concentrated and dialyzed againstdialysis buffer (20 mM Iris-HCl, 2 mM TCEP) overnight. The protein wasaliquoted and frozen at −80° C. until further use.

Sirtuin-modulating compounds of Formula (I) that activated SIRT1 wereidentified using the assay described above and are shown below inTable 1. The EC_(1.5) values represent the concentration of testcompounds that result in 150% activation of SIRT1. The EC_(1.5) valuesfor the activating compounds of Formula (I) are represented by A(EC_(1.5)<1 μM), B (EC_(1.5) 1-25 μM), C (EC_(1.5)>25 μM). The percentmaximum fold activation is represented by A (Fold activation ≧150%) or B(Fold Activation <150%). “NT” means not tested; “ND” means notdeterminable. The compound numbering in the table starts with compoundnumber 10, and parenthetic numbering (#) corresponding to the STACnumbering system in FIG. 4 and Examples 90-106 (i.e., compound no. 68 isalso STAC 1, so it is shown as 68(1), and further STACs: 546(3), 444(4),314(5), 816(7), 76(8), and 81(9)).

TABLE 1 Compounds of Formula (I). Tryp Tryp Com- AVG % pound EC_(1.5)Fold No. [M + H]+_([calc]) Structure (μM) Act  10 427

A A  11 427

A A  12 427

A A  13 454

B A  14 440

C B  15 444

A A  16 513

A A  17 426

A A  18 517

A A  19 392

A A  20 444

A A  21 440

B A  22 487

B A  23 427

A A  24 410

B A  25 429

A A  26 531

B A  27 441

A A  28 441

A A  29 441

A A  30 468

A A  31 501

A A  32 471

A A  33 474

A A  34 457

A A  35 454

B A  36 443

A A  37 454

B A  38 441

A A  39 458

A A  40 443

A A  41 471

A A  42 458

A A  43 410

B A  44 406

B A  45 458

A A  46 444

A A  47 457

A A  48 460

A A  49 429

A A  50 423

B A  51 391

B A  52 405

C B  53 516

A A  54 483

A A  55 497

B A  56 446

A A  57 376

A A  58 377

B A  59 394

A A  60 407

B A  61 440

A A  62 390

B A  63 483

A A  64 481

B A  65 467

B A  66 482

A A  67 509

C B  68  (1) 492

A A  69 467

C B  70 481

C B  71 481

C B  72 488

B A  73 466

B A  74 394

B A  75 488

A A  76  (8) 491

A A  77 470

B A  78 503

B A  79 517

B A  80 486

A A  81  (9) 493

A A  82 443

B A  83 477

A A  84 490

C B  85 442

B A  86 491

A A  87 479

A A  90 482

A A  91 481

B A  92 443

B A  93 481

A A  94 509

B A  95 497

A A  96 460

B A  98 433

B A  99 432

A A 100 446

B A 101 393

A A 102 426

A A 103 453

B A 104 423

B A 105 420

B A 106 406

C A 107 392

A A 108 395

B A 109 395

B A 110 393

A A 114 442

B A 118 445

A A 119 410

A A 120 393

A A 121 423

A A 122 392

A A 123 444

C B 124 423

B A 128 445

A A 129 460

A A 130 401

A A 131 445

B A 132 458

B A 133 458

A A 134 457

C B 135 473

A A 136 427

A A 137 426

C B 138 442

A A 139 383

A A 140 414

A A 141 424

A A 142 427

A A 143 433

B A 144 450

B A 145 450

A A 146 466

B A 147 401

B A 148 450

B A 149 466

B A 150 445

A A 151 445

A A 152 444

C A 153 460

A A 154 450

B A 155 448

B A 156 479

A A 157 466

B A 158 440

B A 159 456

A A 160 456

A A 161 490

A A 162 508

A A 163 442

A A 164 443

A A 165 476

A A 166 443

A A 167 445

B A 168 471

A A 169 471

B A 170 501

A A 171 441

B A 172 441

B A 173 474

A A 174 460

A A 175 495

A A 176 473

A A 177 445

A A 178 449

A A 179 417

A A 180 383

A A 181 384

A A 182 401

A A 183 414

B A 184 436

A A 185 386

B A 186 386

B A 187 461

B A 188 458

B A 189 384

A A 190 414

B A 191 493

A A 192 427

B A 193 428

B A 194 440

A A 195 440

B A 196 441

B A 197 473

A A 198 445

B A 199 430

B A 200 440

C B 201 460

B A 202 427

B A 203 444

B A 204 410

B A 205 406

B A 206 428

B A 207 480

A A 208 430

B A 209 428

C B 210 397

B A 211 434

B A 212 499

A A 213 461

B A 214 453

A A 215 467

B A 216 476

A A 217 477

B A 218 410

A A 219 388

B A 220 373

A A 221 373

B A 222 374

A A 223 391

A A 224 391

B A 225 397

B A 226 398

B A 227 415

B A 228 415

B A 229 433

B A 230 447

A A 231 461

B A 232 451

B A 233 492

A A 234 393

B A 235 458

A A 236 374

B A 237 391

B A 238 391

B A 239 464

A A 240 404

B A 241 404

B A 242 394

B A 243 408

C A 244 412

B A 245 422

B A 246 395

B A 247 400

B A 248 414

NT NT 249 394

B A 250 424

A A 251 415

C A 252 449

C A 253 433

C B 254 463

A A 255 476

B A 256 494

B A 257 481

B A 258 316

C A 259 334

B A 260 393

A A 261 411

A A 262 438

B A 263 420

B A 264 481

A A 265 446

B A 266 416

B A 267 446

B A 268 446

A A 269 463

B A 270 463

B A 271 481

A A 272 440

B A 273 458

A A 274 471

A A 275 394

A A 276 428

A A 277 445

A A 278 428

A A 279 445

A A 280 445

A A 281 476

A A 282 463

A A 283 463

A A 284 378

B A 285 513

A A 286 447

A A 287 475

B A 288 447

A A 289 461

A A 290 465

A A 291 465

B A 292 448

A A 293 448

A A 294 448

B A 295 453

A A 296 467

B A 297 467

B A 298 481

B A 299 513

A A 300 447

C B 301 475

C B 302 447

B A 303 461

B A 304 465

B A 305 447

B A 306 448

NT NT 307 448

B A 308 448

C A 309 477

B A 310 453

B A 311 467

C B 312 467

B A 313 481

C B 314  (5) 440

A A 315 533

B A 316 427

A A 317 458

B A 318 495

B A 319 496

B A 320 526

B A 321 513

B A 322 482

A A 323 513

B A 324 426

A A 325 373

A A 326 436

B A 327 377

C A 328 437

B A 329 410

A A 330 400

A A 331 362

B A 332 426

A A 333 444

A A 334 444

B A 335 547

A A 336 427

B A 337 428

B A 338 428

B A 339 458

B A 340 445

B A 341 401

A A 342 389

B A 343 415

B A 344 394

A A 345 402

B A 346 389

B A 347 426

A A 348 397

A A 349 362

B A 350 397

A A 351 398

A A 352 398

A A 353 415

A A 354 432

A A 355 463

B A 356 450

A A 357 450

B A 358 408

C B 359 465

A A 360 447

A A 361 464

A A 362 465

C A 363 495

B A 364 427

B A 365 458

A A 366 458

A A 367 496

A A 368 527

B A 369 441

A A 370 362

B A 371 402

B A 372 400

B A 373 384

A A 374 377

B A 375 451

A A 376 451

A A 377 469

A A 378 452

A A 379 452

A A 380 452

A A 381 398

B A 382 495

C B 383 449

A A 384 433

B A 385 446

A A 386 415

B A 387 406

A A 388 464

A A 389 497

A A 390 495

B A 391 514

A A 392 441

A A 393 442

A A 394 472

A A 395 362

B A 396 423

A A 397 395

C A 398 421

B A 399 390

B A 400 378

B A 401 425

A A 402 441

A A 403 416

A A 404 422

A A 405 438

A A 406 407

A A 407 406

A A 408 437

A A 409 406

A A 410 514

A A 411 459

A A 412 442

A A 413 378

C B 414 363

B A 415 437

A A 416 413

C B 417 408

A A 418 452

B A 419 402

A A 420 395

C B 421 424

A A 422 446

A A 423 465

A A 424 477

B A 425 482

A A 426 398

B A 427 475

A A 428 793

A A 429 454

B A 430 807

A A 431 409

A A 432 392

B A 433 473

B A 434 437

C B 435 461

A A 436 447

A A 437 477

B A 438 397

B A 439 415

B A 440 428

C B 441 373

B A 442 404

B A 443 409

B A 444  (4) 468

A A 445 568

A A 446 453

B A 447 437

C B 448 446

B A 449 397

B A 450 398

B A 451 398

B A 452 442

A A 453 391

B A 454 374

B A 455 373

B A 456 374

B A 457 374

B A 458 403

B A 459 385

A A 460 508

B A 461 427

A A 462 447

B A 463 464

A A 464 464

B A 465 387

B A 466 407

B A 467 388

B A 468 374

B A 469 451

C B 470 391

A A 471 425

B A 472 441

A A 473 506

A A 474 464

A A 475 464

A A 476 431

B A 477 405

B A 478 418

B A 479 388

B A 480 439

A A 481 407

A A 482 376

B A 483 426

A A 484 409

C B 485 440

A A 486 390

C A 487 390

B A 488 376

B A 489 376

B A 490 362

B A 491 477

A A 492 458

A A 493 348

B A 494 390

B A 495 393

B A 496 406

A A 497 472

A A 498 373

A A 499 404

A A 500 404

A A 501 404

B A 502 426

A A 503 388

A A 504 387

A A 505 387

C B 506 421

A A 507 421

A A 508 421

C A 509 405

A A 510 405

B A 511 418

C B 512 388

B A 513 388

C A 514 387

B A 515 387

A A 516 387

B A 517 388

A A 518 388

C B 519 388

A A 520 388

C A 521 424

B A 522 437

B A 523 467

A A 524 362

B A 525 376

B A 526 393

A A 527 418

A A 528 488

B A 529 458

B A 530 845

A A 531 376

B A 532 377

B A 533 406

B A 534 407

B A 535 416

B A 536 457

B A 537 491

B A 538 457

A A 539 458

B A 540 518

A A 541 504

A A 542 504

A A 543 407

B A 544 407

B A 545 425

B A 546  (3) 939

B B 547 925

C B 548 564

B A 549 424

B A 550 408

B A 551 407

B A 552 438

C B 553 468

B A 554 441

B A 555 392

B A 556 427

A A 557 398

A A 558 399

B A 559 475

A A 560 458

B A 561 464

B A 562 570

A A 563 857

A A 565 823

NT NT 569 440

B A 570 391

B A 571 409

B A 572 391

B A 573 392

C B 574 457

A A 575 473

A A 576 408

B A 577 425

B A 578 452

B A 579 422

B A 580 492

A A 581 492

A A 582 505

B A 583 522

A A 587 416

B A 588 493

A A 589 493

A A 590 507

A A 591 507

A A 592 594

A A 593 494

A A 594 551

A A 595 507

A A 596 507

A A 597 439

B A 598 453

B A 599 467

B A 600 481

B A 601 442

A A 602 484

B A 603 546

C B 604 649

A A 605 621

A A 606 521

A A 607 860

A A 608 910

C B 609 580

B A 610 580

B A 611 580

A A 612 476

B A 613 454

B A 614 454

B A 615 454

B A 616 462

B A 617 493

A A 618 570

B A 619 529

C B 620 440

B A 621 442

NT NT 622 795

C B 623 506

A A 624 465

B A 625 501

A A 626 483

A A 627 457

B A 628 508

B A 629 524

A A 630 460

A A 631 443

B A 632 471

C A 633 468

B A 634 484

C B 635 536

A A 636 550

A A 637 562

A A 638 621

A A 639 634

A A 640 521

A A 641 517

C B 642 518

A A 643 504

A A 644 473

B A 645 443

A A 646 433

A A 647 461

A A 648 509

A A 649 489

B A 650 501

C B 651 510

A A 652 504

A A 653 508

A A 654 492

A A 655 503

A A 656 534

A A 657 521

A A 658 508

A A 659 468

A A 660 502

A A 661 443

C A 662 509

B A 663 442

A A 664 503

B A 665 508

A A 666 465

A A 667 502

A A 668 467

A A 669 443

B A 670 456

A A 671 467

A A 672 570

A A 673 468

B A 674 468

B A 675 408

B A 676 422

A A 677 426

B A 678 409

C B 679 439

B A 680 469

B A 681 408

B A 682 409

B A 683 474

A A 684 409

B A 685 475

B A 688 442

B A 689 471

B A 690 457

B A 691 461

B A 692 506

A A 693 475

A A 694 443

A A 695 444

B A 696 474

B A 697 477

B A 698 509

A A 699 476

B A 700 444

B A 701 444

B A 702 516

NT NT 703 510

A A 704 504

B A 705 443

B A 706 655

A A 708 992

A A 709 509

A A 710 502

A A 714 481

A A 715 503

B A 716 444

B A 717 499

A A 718 499

A A 719 493

A A 720 494

A A 721 494

A A 722 443

A A 723 503

A A 724 503

A A 725 521

A A 726 510

B B 727 444

A A 730 1013 

A A 731 523

A A 733 510

A A 734 508

B A 736 495

A A 737 509

A A 739 444

A A 740 495

A A 741 528

A A 742 525

A A 743 559

A A 745 511

B A 746 440

A A 747 470

A A 748 440

A A 749 645

A A 750 649

A A 751 645

A A 752 663

A A 753 659

A A 754 663

A A 755 454

B A 756 581

A A 757 468

A A 758 567

A A 759 649

B A 760 659

A A 763 508

A A 764 545

NT NT 765 549

A A 766 545

A A 767 563

A A 768 559

NT NT 769 563

NT NT 770 549

A A 771 559

NT NT 776 563

A A 777 510

A A 780 639

A A 785 425

C B 786 491

B A 788 624

A A 790 510

A A 792 524

A A 793 554

B A 794 487

B A 795 501

B A 796 630

B A 797 519

A A 798 548

A A 799 537

A A 800 587

C B 801 548

A A 802 519

B A 803 490

A A 804 520

A A 805 520

B A 806 472

A A 807 490

C B 808 455

A A 809 520

B A 810 1308 

NT NT 811 1294 

NT NT 812 1473 

NT NT 813 1487 

NT NT 814 1109 

NT NT 815 1123 

NT NT 816  (7) 426

A A 817 426

A A 818 517

A A 819 516

A A 820 440

A A 821 440

A A 822 393

A A 823 426

A A 824 426

A A 825 426

NT NT 826 358

NT NT 827 425

NT NT

In certain embodiments, the compound is any one of Compound Numbers 1,3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 25,27, 28, 29, 30, 31, 32, 33, 34, 36, 38, 39, 40, 41, 42, 45, 46, 47, 48,49, 53, 54, 56, 57, 59, 61, 63, 66, 68, 75, 76, 80, 81, 83, 86, 87, 90,93, 95, 99, 101, 102, 107, 110, 118, 119, 120, 121, 122, 128, 129, 130,133, 135, 136, 138, 139, 140, 141, 142, 145, 150, 151, 153, 156, 159,160, 161, 162, 163, 164, 165, 166, 168, 170, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 184, 189, 191, 194, 197, 207, 212, 214, 216,218, 220, 222, 223, 230, 233, 235, 239, 250, 254, 260, 261, 264, 268,271, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 285, 286,288, 289, 290, 292, 293, 295, 299, 314, 316, 322, 324, 325, 329, 330,332, 333, 335, 341, 344, 347, 348, 350, 351, 352, 353, 354, 356, 359,360, 361, 365, 366, 367, 369, 373, 375, 376, 377, 378, 379, 380, 383,385, 387, 388, 389, 391, 392, 393, 394, 396, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 415, 417, 419, 421, 422, 423, 425,427, 428, 430, 431, 435, 452, 459, 461, 463, 470, 472, 473, 474, 475,480, 481, 483, 485, 491, 492, 496, 497, 498, 499, 500, 502, 503, 504,505, 506, 507, 509, 515, 517, 519, 523, 526, 527, 530, 538, 540, 541,542, 556, 557, 559, 562, 563, 574, 575, 580, 581, 583, 588, 589, 590,591, 592, 593, 594, 595, 596, 601, 604, 605, 606, 607, 611, 617, 623,625, 626, 629, 630, 635, 636, 637, 638, 639, 640, 642, 643, 645, 646,647, 648, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 663, 665,666, 667, 668, 670, 671, 672, 676, 683, 692, 693, 694, 698, 703, 706,708, 709, 710, 714, 717, 718, 719, 720, 721, 722, 723, 724, 725, 727,730, 731, 733, 736, 737, 739, 740, 741, 742, 743, 746, 747, 748, 749,750, 751, 752, 753, 754, 756, 757, 758, 760, 763, 765, 766, 767, 770,776, 777, 780, 788, 790, 792, 797, 798, 799, 801, 803, 804, 806 and 808.

Example 107(4S)—N-(pyridin-3-yl)-7-(4-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide

To a degassed solution of((4S)-7-chloro-N-(pyridin-2-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(700 mg, 2.217 mmol), 4-(trifluoromethyl)piperidine (679 mg, 4.43 mmol)in 1,4-Dioxane (20 mL) was addeddicyclohexyl(2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphine (423mg, 0.887 mmol), potassium carbonate (919 mg, 6.65 mmol) andpalladium(II) acetate (100 mg, 0.443 mmol) subsequentially at 20° C. andthe reaction mixture was stirred in a sealed tube at 90° C. for 16 hr.The reaction mixture was poured in to cold water (70 mL) and extractedwith ethyl acetate (150 mL). The organics were separated and dried overanhydrous sodium sulphate, concentrated under reduced pressure to givethe crude product. The crude product was added to a silica gel columnand was eluted with 1% to 2% methanol in dichloro methane. Collectedfractions to give 500 mg, It was again purified by GRACE reverse phaseHPLC to give(4S)—N-(pyridin-3-yl)-7-(4-(trifluoromethyl)piperidin-1-yl)-3,4-dihydro-1,4-methanopyrido[2,3-b][1,4]diazepine-5(2H)-carboxamide(320 mg, 0.710 mmol, 32%). MS (ESI) calcd for C₂₁H₂₃F₃N₆O: 433.2.

EQUIVALENTS

The present invention provides among other things sirtuin-modulatingcompounds and methods of use thereof. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) (www.tigr.org) and/or theNational Center for Biotechnology Information (NCBI)(www.ncbi.nlm.nih.gov).

We claim:
 1. A compound which is:

or a pharmaceutically acceptable salt thereof.
 2. A compound which is:


3. A pharmaceutical composition comprising a compound of claim 1 orpharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable excipient, carrier or diluent.
 4. A pharmaceuticalcomposition comprising a compound of claim 2 and a pharmaceuticallyacceptable excipient, carrier or diluent.